Matrices for drug delivery and methods for making and using the same

ABSTRACT

In one aspect, biocompatible matrices such as sol-gels encapsulating a reaction center may be administered to a subject for conversion of prodrugs into biologically active agents. In certain embodiments, the biocompatible matrices of the present invention are sol-gels. In one embodiment, the enzyme  L -amino acid decarboxylase is encapsulated and implanted in the brain to convert  L -dopa to dopamine for treatment of Parkinson&#39;s disease.

1. RELATED APPLICATION INFORMATION

This Application claims the benefit of priority under 35 U.S.C. section119(e) to Provisional Application 60/119,828, filed Feb. 12, 1999.

2. INTRODUCTION

Many clinical conditions, deficiencies, and disease states may beremedied or alleviated by providing to a patient beneficial biologicallyactive agents or removing from the patient deleterious biologicallyactive agents. In many cases, provision of beneficial agents or removalof deleterious ones may restore or compensate for the impairment or lossof function or homeostasis. An example of a disease or deficiency statewhose etiology includes loss of such an agent include Parkinson'sdisease, in which dopamine production is diminished. The impairment orloss of such agents may result in the loss of additional metabolicfunctions.

Parkinson's disease, one of many motor system disorders, results insymptoms such as tremor, bradykinesia, and impaired balance. Keller inHandbook of Parkinson's Disease (Marcel-Dekker Inc.: New York 1992).Parkinson's disease is both chronic and progressive, and nearly 50,000Americans are diagnosed with Parkinson's disease each year. More thanhalf a million Americans are currently being treated for Parkinson'sdisease. Bennett et al. Dis. Mon. 38:1 (1992).

A specific area of the brain known as the basal ganglia is affected inParkinson's disease. The basal ganglia plays a vital role in voluntarymovement control. A region of the basal ganglia termed the substantianigra is important in the synthesis of the neurotransmitter dopamine.Deterioration of the dopamine producing cells in the substantia nigraresults in the characteristic symptoms of Parkinson's disease. Thesesymptoms are thought to be due to a deficiency of dopamine in both thesubstantia nigra and the striatum. Obeso et al., Advances in Neurology74:143 (1997). The striatum requires a balance of the neurotransmittersdopamine and acetylcholine in order to control properly movement,balance, and walking. The cause of the impairment or death of the cellsresponsible for the production of dopamine in the substantia, althoughcurrently unknown, has been attributed to a number of factors, includingoxidant stress, mitochondrial toxicity, and autoimmunity. Olanow et al.,in Neurodegenetaion and Neuroprotection (Academic Press: San Diego1996).

There are currently a number of methods being used for treatingParkinson's disease, which can be grouped into two categories, namelychemical and surgical methods. Yahr et al. Advances in Neurology 60:11-17 (1993). In chemical treatment methods, the goal is to achieve astasis between the counterbalancing dopamine and acetylcholineneurotransmitters. Jankovic et al., in Parkinson's Disease and MovementDisorders 115-568 (Williams and Wilkins: Baltimore). The correct balanceof the neurotransmitters produces a therapeutic effect in theParkinson's disease patient. At least three methods of accomplishing orrestoring a therapeutic balance are presently possible. First, in thedopaminergic method, a balance may be achieved by increasing deficientdopamine levels by using dopamine precursors or by increasing levels ofdopamine agonists in the brain. Controlled release systems have beenused to increase dopamine levels. Becker et al. Brain Res. 508:60(1990); Sabel Advances in Neurology, 53:513-18 (1990). Second, monoamineoxidase inhibitors (MAO) reduce the rate of dopamine breakdown catalyzedby monoamine oxidase enzymes and thereby increase the dopamine levels inthe brain. Third, anticholinergics block the receptor sites foracetylcholine in an attempt to compensate for low dopamine levels.

Currently, there are at least two surgical methods being utilized inParkinson's therapy. Jankovic et al., supra. In ablative surgeries, asmall portion of the globus pallidus (pallidotomy) or the thalamus(thalamotomy) is destroyed, which has been shown to be effective intreating Parkinson's disease. In tissue transplants, dopaminergic cells,such as fetal nigral primordia and adrenal chromaffin cells, are graftedinto the basal ganglia region or striatum. Fetal dopaninergic neuronshave been observed to provide superior functional recovery in terms ofboth magnitude and duration of effects. Kordower et al., in TherapeuticApproaches To Parkinson's Disease 443-72 (Roller et al. eds., MercerDekker Inc.: New York (1990)). This is true for both rodent and nonhumanprimate models of Parkinson's disease as well as clinical trials inParkinson's disease patients. Bakay et al. Ann. NY Acad. Sci. 495:623-40(1987); Bankiewiez et al. Progress in Brain Research 78:543-50 (1988);Freed et al. New England Journal of Medicine 327:1549-55 (1992). Inaddition, such cells have been encapsulated, Emerich et al. Neurosci.Behav. Rev. 16:437-47 (1992), and found to alleviate symptoms ofParkinson's disease in rodents, Aebischer et al. Brain Res. 560:43(1991); Lindner et al. Exp. Neurol. 132:62-76 (1995); Subramanian et al.Cell Transplant 6:469-77 (1997).

Although both chemical and surgical methods help to decrease thesymptoms of Parkinson's disease, there are a number of areas requiringimprovement. With respect to chemical methods, delivery to the striatalregion of any biologically active agent, such as dopamine, MAOinhibitors, or anticholinergics, is complicated, in part, because of thepresence of the blood-brain barrier, which may result in lowbioavailability of any such agents. As an alternative, directadministration of dopamine into the central nervous system may requirethe frequent and repeated use of invasive procedures which compromisethe integrity of the blood-brain barrier. Those techniques requirerepeated infusions into the brain, either through injections viacannulae, or from pumps which must be replaced every time the reservoiris depleted. Even with the careful use of sterile procedures, there isrisk of infection. It has been reported that even in intensive careunits, intracerebroventricular catheters used to monitor intracranialpressure become infected with bacteria after about three days. Saffran,Perspectives in Biology and Medicine 35:471-86 (1992). In addition tothe risk of infection, there seems to be some risk associated with theinfusion procedure. Infusions into the ventricles have been reported toproduce hydrocephalus, Saffran et al. Brain Research 492:245-54 (1989),and continuous infusions of solutions into the parenchyma is associatedwith necrosis.

Because of the fact that dopamine itself does not readily cross theblood-brain barrier, many of the drug therapies utilize the dopamineprecursor L-dopa. Modern Pharmacology 108 (2d ed, Craig et al. eds,1986). Conversion of L-dopa to dopamine requires the enzyme amino aciddecarboxylase, which is found in the substantia nigra of the brain. Theprogression of Parkinson's disease and the need for larger doses ofL-dopa in order to produce therapeutic effects may be due to the loss ofthe enzyme required for this conversion. This loss of therapeuticefficacy is known as long-term L-dopa syndrome and occurs in 3 to 5years in 50% of Parkinson's disease patients being treated with L-dopa.Brannan et al. Neurology 45:596 (1991).

Surgical tissue transplantation suffers from a number of factors such asimmunogenic complications, delayed improvement results, and low tissuesurvival rates of around 10%. The use of fetal tissue has formidablehurdles, including the failure to reestablish the normal neuralcircuitry, high mortality and morbidity associated with the transplantprocedure, and the ethical issue of human fetal tissue research.Aebischer et al. Transactions of the ASME 113:178 (1991). Adrenal cellsare generally only implanted in patients less than 60 years of age, asthe adrenal gland of older patients may not contain sufficientdopamine-secreting cells, which limits the usefulness of the procedureas a treatment method because the disease most often affects theelderly. With respect to encapsulation of dopamine producing cells,questions remain concerning cell viability upon encapsulation and theirresulting durability and output. Lindner et al. Cell Transplant 7:165-74(1998).

Although the different therapies discussed above for Parkinson's diseasehave met with some success, there remains a need for additionaltreatment methods for the condition. In the present invention, in part,novel methods of producing the biologically active agent dopamine in thebrain is contemplated. In another aspect, the present inventioncontemplates treating diseases or conditions by either producing orremoving biologically active agents in a patient.

3. SUMMARY OF THE INVENTION

In one aspect, the present invention contemplates matrices encapsulatinga reaction center, and methods of using the same.

In another aspect, the present invention is directed to methods forproducing a biologically active agent from a prodrug involvingencapsulating a cell-free reaction center in a biocompatible matrix andadministering the matrix to a subject, wherein said reaction centerconverts a prodrug into a biologically active agent in the subject. Inone method of the present invention, the matrices of the presentinvention are administered to a subject for treatment of a disease orcondition by production or removal of a biologically active agent oragents.

In another aspect, the present invention involves methods of enzymereplacement therapy for treating a subject involving administering tothe subject a reaction center which is encapsulated in a biocompatiblematrix, wherein said reaction center replaces, augments, or supplementssome activity in said subject. The reaction center may be an enzyme inwhich a subject to be treated is deficient, because of, for example, adisease or condition or an inborn error of metabolism.

In another aspect, the present invention contemplates methods for theextra-corporeal use of the subject matrices in, for example, organassist devices such as a liver assist device. In one method of thepresent invention, the matrices of the present invention are used exvivo for treatment of a disease or condition by production or removal ofa biologically active agent or agents from a patient.

In certain embodiments of the present invention, including the foregoingaspects, the reaction center may be an enzyme, an antibody, a catalyticantibody or other biological material. In other embodiments, the matrixmay be an inorganic-based sol-gel matrix or a silica-based sol-gelmatrix. More than one reaction center may be encapsulated in a singlematrix. In addition to any encapsulated reaction center, the matrix mayhave encapsulated additives. In one preferred embodiment, the reactioncenter may be L-amino acid decarboxylase, the prodrug may be L-dopa andthe biologically active agent may be dopamine.

In still another aspect, the matrices of the present invention, andmethods of using the same, may be used in diagnostic applications, suchas in certain embodiments in which an imaging agent is encapsulatedtherein.

In still another aspect, the matrices and compositions of the presentinvention may be used in the manufacture of a medicament for any numberof uses, including for example treating any disease or other treatablecondition of a patient. In still other aspects, the present invention isdirected to a method for formulating (either separately or together)matrices, prodrugs and other materials and agents required for treatmentin a pharmaceutically acceptable carrier.

In another aspect, this invention contemplates a kit including matricesof the present invention, and optionally instructions for their use. Forexample, in one embodiment, such kits include matrices and associatedprodrug for treatment of a patient. Such kits may have a variety ofuses, including, for example, imaging, diagnosis, therapy, vaccination,and other applications.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Enzymatic activity assay for a matrix containing β-Glucosidase.

FIG. 2: Substrate and product spectra for penicillinase assay.

FIGS. 3(a) and (b): Penicillinase activity assays showing (a) multipleassays of a single matrix and (b) a single assay performed on each offive matrices from one batch preparation.

FIG. 4: Change in absorbance at three hours as a function of the enzymeconcentration added to the matrix during preparation.

FIG. 5: Yield of immobilized enzyme in penicillinase-containing sol-gelmatrices (observed activity was calculated as the percentage of enzymeactivity used in the preparation of the matrices).

FIG. 6. Activity of crushed and whole matrices containing penicillinase.FIGS. 6 and 7 both show data for five unique matrices assayed one timeeach.

FIG. 7: Penicillinase activity in whole monoliths and crushed matriceswith points shown being the mean of five measurements (error bars+/−onestandard deviation).

FIGS. 8(a) and (b): (a) Activity of penicillinase-containing matriceswith varying surface areas, and (b) activity as a percentage of theactivity added in preparation. Surface areas corresponding to thelabeling in the graphs are: A=15.1 cm², B=39.2 cm², C=71.3 cm² and D=135cm².

FIG. 9: Tyrosine decarboxylase activity assay; elapsed time since gelcast 19.5 h.

FIG. 10. Tyrosine decarboxylase activity assay of two identical 16 dayold matrices, A and B, with comparison to C (same matrix compositionaged 19 h, no cofactor present). Assay of A is performed in the absenceof pyridoxal-5-phosphate (cofactor) while B is performed with cofactorpresent.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Definitions

For convenience, the meanings of certain terms employed in thespecification are provided below. The meanings for these terms, as thoseof skill in the art would understand them, should be read in light ofthe remainder of the specification for a full and complete understandingof the scope of the invention.

The term “additives” refers to compounds, materials, and compositionsthat may be included in a matrix along with a reaction center. Anadditive may be encapsulated in or on a matrix or attached to a matrix,either the interior or exterior, by some interaction, including acovalent one or adhesion of the additive to the matrix. Examples ofadditives include other molecules necessary for the conversion mediatedby the reaction center, solid materials which serve as a framework forthe matrix, etc.

The term “antibody” refers to a binding agent including a whole antibodyor a binding fragment thereof which is reactive with a specific antigen.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)2 fragments can be generated bytreating an antibody with pepsin. The resulting F(ab)2 fragment can betreated to reduce disulfide bridges to produce Fab fragments.

The term “biocompatible matrix” as used herein means that the matrix,upon implantation in a subject, does not elicit a detrimental responsesufficient to result in the rejection of the matrix or to render itinoperable, for example through degradation. To determine whether anysubject matrix is biocompatible, it may be necessary to conduct atoxicity analysis. Such assays are well known in the art. Onenon-limiting example of such an assay for analyzing a composition of thepresent invention would be performed with live carcinoma cells, such asGT3TKB tumor cells, in the following manner: various amounts of subjectmatrices are placed in 96-well tissue culture plates and seeded withhuman gastric carcinoma cells (GT3TKB) at 104/well density. The degradedproducts are incubated with the GT3TKB cells for 48 hours. The resultsof the assay may be plotted as % relative growth versus amount ofmatrices in the tissue-culture well. In addition, matrices of thepresent invention may also be evaluated by well-known in vivo tests,such as subcutaneous implantations in rats to confirm that they do notcause significant levels of irritation or inflammation at thesubcutaneous implantation sites.

The term “biologically active agent” as used herein means any organic orinorganic agent that is biologically active, e.g., produces somebiological affect in a subject.

The term “encapsulated reaction center” means a reaction center that iscontained within or on a matrix. For example, an encapsulated reactioncenter may be immobilized somewhere in a silica matrix; alternatively,it may be attached to the interior or the surface of a matrix by somemeans other than physical confinement, such as by covalent bonds oradhesion. Alternatively, an encapsulated reaction center may be locatedon the surface of a matrix.

The term “enzyme” refers to any polypeptide that converts a prodrug intoa biologically active agent. An enzyme may be isolated from naturallyoccurring sources, or it may be prepared by recombinant methods. Anenzyme may be a fusion or chimeric protein of a polypeptide thatconverts a prodrug and another polypeptide. An enzyme may be a portionor a fragment of a full-length enzyme. An enzyme may be substantiallypurified, or only partially purified. Homologs, orthologs, and paralogsof an enzyme are also enzymes. For purposes of the present invention, anenzyme is not a catalytic antibody, a cell, or an organism.

“Homology” refers to sequence similarity between two polypeptides orbetween two nucleic acid molecules. Homology may be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. An“unrelated” or “non-homologous” sequence shares less than 40 percentidentity, though preferably less than 25 percent identity, with thesequence to which it is being compared.

The term “immunoisolatory matrix” means that the matrix uponadministration to subject minimizes the deleterious effects of thesubject's immune system on the reaction center or other contentscontained within the matrix.

The term “long-term, stable production of biologically active agent” asused herein means the continued production of a biologically activeagent at a level sufficient to maintain its useful biological activityfor periods greater than at least about one month, more preferably abouttwo months, four months, six months, eight months, ten months, one year,one and a half years or more.

The term “matrix” means any material in which a reaction center has beenencapsulated. For example, one type of matrix is a silica-based sol-gelmatrix. Another example of a matrix is an inorganic-based sol-gelmatrix. A matrix may have more than one type of reaction centerencapsulated.

The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

The phrases “parenteral administration” and “administered parenterally”mean modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal and intrasternal injection and infusion.

A “patient” or “subject” to be treated by the present invention can meaneither a human or non-human animal.

The phrase “pharmaceutically acceptable” is employed to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a prodrug, compound,material, or composition from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical formulations.

The term “prodrug” is intended to encompass compounds, materials, andcompositions which are converted by an encapsulated reaction center intoa biologically active agent. One means of converting a prodrug to abiologically active agent is by an enzyme-catalyzed reaction. A prodrugneed not be biologically inactive itself; instead, to be a prodrug, acompound need only have some altered biological activity upon conversionby a reaction center. A prodrug may be either endogenous or exogenous toa subject. Also, many prodrugs produce more than one compounds uponcertain types of conversion, and the term “biologically active agent”when used to refer the products of such a prodrug conversion is intendedto encompass all of those products.

The term “reaction center” means any material or compound that may beencapsulated in a matrix and that converts or reacts a prodrug into abiologically active agent or reacts with a biologically active agent to,for example, degrade such agent. In certain embodiments, the reactioncenter may be an enzyme, a catalytic antibody, or a nonbiologicallyderived catalyst, such as those commonly used for organic synthesis. Incertain embodiments, the reaction center may be prokaryotic oreukaryotic cells, such as bacteria, yeast, or mammalian cells, includinghuman cells, or components thereof, such as organelles. In otherembodiments of the present invention, the reaction center issubstantially pure, i.e. 95%, 96%, 97%, 98% or 99% pure and thereforeessentially cell-free or organism-free. Numerous examples of reactioncenters are set forth below.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” mean theadministration of a compound, drug or other material other than directlyinto the central nervous system such that it enters the patient's systemand, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

The phrase “therapeutically effective amount” means that amount of aprodrug, biologically active agent, compound, material, or compositionaccording to the present invention which is effective for producing somedesired therapeutic effect. Because in certain embodiments of thepresent invention, a prodrug is converted into a biologically activeagent by an encapsulated reaction center, it is necessary to considerthis conversion in determining what may be a “therapeutically effectiveamount” of a prodrug. The amount can vary greatly according to theeffectiveness of a matrix, prodrug, or biologically active agent, theage, weight, and response of the individual subject, as well as thenature and severity of the subject's symptoms. Accordingly, there is noupper or lower critical limitation upon the amount of the a matrix,prodrug, or biologically active agent. The required quantity to beemployed of a matrix or prodrug in combination with a matrix in thepresent invention may readily be determined by those skilled in the art.

The terms “treating” or “method of treatment” (and variations thereof)is intended to encompass curing as well as ameliorating at least onesymptom of a condition, deficiency, or disease.

The term “ED₅₀” means the dose of a drug, including, for example, amatrix or a combination of a matrix and prodrug, which produces 50% of amaximum response or effect. Alternatively, the dose which produces apre-determined response in 50% of test subjects or preparations.

The term “LD₅₀” means the dose of a drug, including, for example, amatrix or a combination of a matrix and prodrug, which is lethal in 50%of test subjects.

The term “therapeutic index” refers to the therapeutic index of a drug,including, for example, a matrix or a combination of a matrix andprodrug, defined as LD₅₀/ED₅₀.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,alkylaminos, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The term “aryl” refers to both substituted andunsubstituted aromatic rings. The aromatic ring can be substituted atone or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromaticor heteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocycle” refer to 4- to 10-memberedring structures, more preferably 3- to 7-membered rings, whose ringstructures include one to four heteroatoms. Heterocycles can also bepolycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, quinoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The phrase “fused ring” is art recognized and refers to a cyclic moietywhich can comprise from 4 to 8 atoms in its ring structure, and can alsobe substituted or unsubstituted, (e.g., cycloalkyl, a cycloalkenyl, anaryl, or a heterocyclic ring) that shares a pair of carbon atoms withanother ring. To illustrate, the fused ring system can be abenzodiazepine, a benzoazepine, a pyrrolodiazepine, a pyrroloazepine, afuranodiazepine, a furanoazepine, a thiophenodiazepine, athiophenoazepine, an imidazolodiazepine, an imidazoloazepine, anoxazolodiazepine, an oxazoloazepine, a thiazolodiazepine, athiazoloazepine, a pyrazolodiazepine, a pyrazoloazepine, apyrazinodiazepine, a pyrazinoazepine, a pyridinodiazepine, apyridinoazepine, a pyrimidinodiazepine, or a pyrimidinoazepine.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈₀, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈₀ represents an aryl, a cycloalkyl,a cycloalkenyl, a heterocycle or a polycycle; and m is zero or aninteger in the range of 1 to 8. In preferred embodiments, only one of R₅or R₁₀ can be a carbonyl, e.g., R₅, R₁₀ and the nitrogen together do notform an imide. In even more preferred embodiments, R₉ and R₁₀ (andoptionally R′₁₀) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R₈₀. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₉ and R₁₀is an alkyl group.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈₀, where m and R₈₀ are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈₀, wherein m and R₈₀ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈₀ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈₀, where m and R₉₀ are as defined above.Where X is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R₁₁ is as definedabove, the moiety is referred to herein as a carboxyl group, andparticularly when R₁₁ is a hydrogen, the formula represents a“carboxylic acid”. Where X is an oxygen, and R′₁₁ is hydrogen, theformula represents a “formate”. In general, where the oxygen atom of theabove formula is replaced by sulfur, the formula represents a“thiolcarbonyl” group. Where X is a sulfur and R₁₁ or R′₁₁ is nothydrogen, the formula represents a “thiolester.” Where X is a sulfur andR₁₁ is hydrogen, the formula represents a “thiolcarboxylic acid.” WhereX is a sulfur and R₁₁′ is hydrogen, the formula represents a“thiolformate.” On the other hand, where X is a bond, and R₁₁ is nothydrogen, the above formula represents a “ketone” group. Where X is abond, and R₁₁ is hydrogen, the above formula represents an “aldehyde”group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R₈₀, where m and R₈₀ are described above.

The terms “sulfoxido”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R′₁₁ is as defined above, but is not hydrogen.

A “sulfone”, as used herein, refers to a moiety that can be representedby the general formula:

in which R′₁₁ is as defined above, but is not hydrogen.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

A “phosphoramidate” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphonamidate” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, etc., when itoccurs more than once in any structure, is intended to be independent ofits definition elsewhere in the same structure.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivitization with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts are formed withan appropriate optically-active acid or base, followed by resolution ofthe diastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the desired use ofthe compound.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, hydrolysis, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

By the terms “amino acid residue” and “peptide residue” is meant anamino acid or peptide molecule without the —OH of its carboxyl group(C-terminally linked) or the proton of its amino group (N-terminallylinked). In general the abbreviations used herein for designating theamino acids and the protective groups are based on recommendations ofthe IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry11:1726-1732 (1972)). For instance Met, Ile, Leu, Ala and Gly represent“residues” of methionine, isoleucine, leucine, alanine and glycine,respectively. By the residue is meant a radical derived from thecorresponding α-amino acid by eliminating the OH portion of the carboxylgroup and the H portion of the α-amino group. The term “amino acid sidechain” is that part of an amino acid exclusive of the —CH(NH₂)COOHportion, as defined by Kopple, Peptides and Amino Acids 2, 33 W. A.Benjamin Inc., New York and Amsterdam, 1966; examples of such sidechains of the common amino acids are —CH₂CH₂SCH₃ (the side chain ofmethionine), —CH(CH₃)—CH₂CH₃ (the side chain of isoleucine),—CH₂CH(CH₃)₂ (the side chain of leucine) or H— (the side chain ofglycine).

For the most part, the amino acids used in the application of thisinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids which contain amino and carboxyl groups. Particularly suitableamino acid side chains include side chains selected from those of thefollowing amino acids: glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan. However, the term amino acidresidue further includes analogs, derivatives and congeners of anyspecific amino acid referred to herein. For example, the presentinvention contemplates the use of amino acid analogs wherein a sidechain is lengthened or shortened while still providing a carboxyl, aminoor other reactive precursor functional group for cyclization, as well asamino acid analogs having variant side chains with appropriatefunctional groups. For instance, such amino acid analogs includeβ-cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine,homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan,1-methylhistidine, or 3-methylhistidine. Other naturally occurring aminoacid metabolites or precursors having side chains which are suitableherein will be recognized by those skilled in the art and are includedin the scope of the present invention.

Also included are the D and L stereoisomers of such amino acids when thestructure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols D, L or DL, furthermore when theconfiguration is not designated the amino acid or residue can have theconfiguration D, L or DL. It will be noted that the structure of some ofthe compounds of this invention includes asymmetric carbon atoms. It isto be understood accordingly that the isomers arising from suchasymmetry are included within the scope of this invention. Such isomersare obtained in substantially pure form by classical separationtechniques and by sterically controlled synthesis. For the purposes ofthe present invention, unless expressly noted to the contrary, a namedamino acid shall be construed to include both the D or L stereoisomers,preferably the L stereoisomer.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed. (Greene et al., ProtectiveGroups in Organic Synthesis, 2^(nd) ed.; Wiley: N.Y., 1991).

The phrase “N-terminal protecting group” or “amino-protecting group” asused herein refers to various amino-protecting groups which can beemployed to protect the N-terminus of an amino acid or peptide againstundesirable reactions during synthetic procedures. Examples of suitablegroups include acyl protecting groups such as, to illustrate, formyl,dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl;aromatic urethane protecting groups as, for example, carbonylbenzyloxy(Cbz); and aliphatic urethane protecting groups such ast-butyloxycarbonyl (Boc) or 9-Fluorenylmethoxycarbonyl (FMOC).

The phrase “C-terminal protecting group” or “carboxyl-protecting group”as used herein refers to those groups intended to protect a carboxylicacid group, such as the C-terminus of an amino acid or peptide. Benzylor other suitable esters or ethers are illustrative of C-terminalprotecting groups known in the art.

5.2. Uses

As a general introduction to the present invention, one way toappreciate certain aspects of the invention is by considering some ofthe different uses to which certain embodiments may be put. Therendition of these uses is for illustrative purposes only, and suchcategorization is not intended to limit the scope of the presentinvention. Other uses may be readily apparent to those of skill in theart, and other features of the present invention are presented below andmay be applicable to any of the following uses. For all these uses,certain embodiments of the present invention may be used in animalmodels for assaying new treatments, e.g., new therapeutics or newtreatment regimes.

5.2.1. Prodrug Activation

In one aspect of the present invention, embodiments of the presentinvention may be used as prodrug activators, that is, for prodrugactivation. When used in this fashion, the reaction center encapsulatedin a matrix reacts with a prodrug or prodrugs to produce a biologicallyactive agent or agents. The prodrug may be exogenous to the subject,thereby requiring administration of the prodrug, or the prodrug may beendogenous to the subject, in which case administration of the prodrugto a subject may be used to add to the prodrug present in the subject,but is not absolutely necessary. For prodrug activation, one featureconcerns matching the prodrug of interest with the reaction centerencapsulated so that the reaction center may convert the prodrug into abiologically active agent. In general, the therapeutic effect of amatrix used for pending activation may vary greatly with its site ofadministration.

One related example of prodrug activation using exogenous sourcesinvolves ADEPT technology, or antibody directed enzymatic prodrugtherapy, whereby an enzyme that converts a prodrug into a cytotoxicagent is attached to an antibody that is targeted to a antigen ofinterest, often a surface cell receptor of a neoplastic growth. Seegenerally Denny et al. J. Pharm. Pharmacol. 50:387-94 (1998) and U.S.Pat. Nos. 6,015,556, 6,005,002, and 5,985,281. After binding of theantibody-enzyme conjugate to the antigen, prodrug is administered. Theenzyme converts the exogenous prodrug into the cytotoxic agent in thevicinity of the neoplastic growth. In this fashion, ADEPT technologyreduces toxicity to the subject because the cytotoxic agent is onlyproduced in the immediate vicinity of the tumor. In gene-directed enzymeprodrug therapy (GDEPT), the exogenous enzyme is generated selectivelyin the tumor cells after delivery of a DNA construct containing thecorresponding gene. The present invention contemplates relying onbystander effects and the like in the same fashion by administration,e.g., implantation, of the matrix in the vicinity of any neoplasm,whereupon administration of a prodrug causes conversion of that prodruginto a biologically active agent adjacent to the neoplasm. In contrastto ADEPT, the matrix may not be cleared from the subject as is oftenobserved for the antibody-enzyme conjugate, so multiple administrationsof prodrug using the present invention may be feasible. The presentinvention may also not result in nonspecific activation of the prodrug,which may occur using ADEPT if the antibody-enzyme conjugated bindsindiscriminately or is not cleared by treatment with another antibodyprior to administration of the prodrug.

Another illustrative report of prodrug activation using a exogenoussource involved localized generation of 5-flurouracil from5-fluorocytosine by surgically implanting immobilized cytosine deaminaseadjacent to subcutaneous tumors in rats and injecting intraperitomeally5-fluorocytosine. Nishiyama et al., Cancer Res. 45:1753-61 (1985). Inthis example, the implanted enzyme was encapsulated in a dialysis tube.

Embodiments of the present invention that may be grouped under thiscategory involve the production of dopamine by a matrix, to which anexample described below is directed. The biosynthesis of dopamineinvolves a number of enzymatic steps. One traditional treatment ofParkinson's disease uses an immediate precursor of dopamine, L dopa.Although L dopa therapy is effective in reducing Parkinson's symptoms,there is a lose of L dopa efficacy over time. One possible means ofovercoming such a loss of efficacy involves increasing the enzymaticactivity necessary to convert L dopa to dopamine. One enzyme that may beused in this regard is aromatic L-amino acid decarboxylase (AADC, E.C.4.1.1.28). Cells stably expressing AADC have been grafted into6-hydroxy-dopamine denervated rat striatum, and upon administration of Ldopa, the dopamine content was observed to increase. Kaddis et al. J.Neurochem. 68:1520-26 (1997).

AADC catalzyes the irreversible decarboxylation reaction of severalaromatic L-amino acids, including L-dopa, m-tyrosine, p-tyrosine,phenylalanine, 5-hydroxytrptophan, and tryptophan. Hayashi et al.Biochemistry 32:812-18 (1993); Dominici et al. Eur. J. Biochem.169:209-13 (1987); Voltattomi et al. Methods in Enzymology, 142:179-87(1987); Sourkes, Methods in Enzymology, 142:170-87 (1987); Lindstrom,Biochem Biophys. Acta 884:276-81 (1986); Jung Bioorganic Chem. 14:429-43(1986); Nishigaki Biochem. J. 252:331-35 (1988). AADC is a pyridoxalphosphate dependent enzyme and is produced in the substantia nigralcells. Nigrastriatal cell death and concomitant decrease in AADCactivity may result in decreased dopamine levels in the brain.Encapsulation of AADC as the reaction center and appropriateadministration may allow for production of dopamine from L-dopa in thenigrastriatal region for treatment of Parkinson's disease.

It is possible that, by using this embodiment or related ones of thepresent invention, it may not be necessary to administer L-dopa toproduce a therapeutic effect. Encapsulated enzymes may not requireexogenous prodrug, e.g., administration of L-dopa, because L-dopa levelsthat occur naturally in a subject may be a sufficient source of dopamineupon conversion by the matrix. As a result, side effects of L-dopaadministration common in present therapeutic treatments, includingnausea, may be avoided. L-dopa therapy presently requires simultaneousadministration of a peripheral decarboxylase inhibitor, such ascarbidopa or benserazide, to reduce decarboxylation of L-dopa todopamine outside the brain, which causes such nausea. Calne et al. NewEng. J. Med. 329:1021-27 (1993).

Although AADC is the enzyme responsible for L-dopa conversion todopamine within the substantia nigra region of the brain, at least oneadditional enzyme is capable of effecting this conversion. L-Tyrosinedecarboxylase (TD, E.C. 4.1.1.25) catalyzes the removal of the carboxylgroup from tyrosine to produce tyramine and carbon dioxide. Pyridoxal5′-phosphate is a necessary coenzyme. Although TD has greaterspecificity for decarboxylation of L-tyrosine to tyramine, TD alsocatalyzes decarboxylation of L-dopa. Maraques et al. Plant Physiol, 88:46-51 (1988). Accordingly, TD may be encapsulated as the reaction centerin a matrix of the present invention as a treatment method forParkinson's disease.

In another approach to Parkinson's treatment using an embodiment of thepresent invention, an encapsulated reaction center may be used toproduce a precursor of dopamine, such as L-dopa. Such a matrix would besimilar to traditional L-dopa treatments, but in this case, L-dopa wouldbe produced by the matrix only in the location where the matrix wasadministered. In this fashion, the matrix could be implanted so thatL-dopa would be produced only in the brain. Consequently, there wouldprobably be no side effects as reported for traditional L-dopa therapy.Tyrosine is converted to L-dopa by the enzyme tyrosine monooxygenase(TMO, E.C. 1.14.16.2). Encapsulation of TMO in a matrix andadministration to the brain should increase L-dopa levels there.Tyrosine readily crosses the blood-brain barrier and would result invery little systematic toxicity. Tyrosine may or may not be necessary toadminister to a subject to achieve the desired therapeutic effect.

In another embodiment of the present invention, it is possible toencapsulate both TMO and either AADC, TD, or both, in a matrix. By doingso, it would be possible for a single matrix to convert tyrosine toL-dopa, and L-dopa to dopamine. Because the decarboxylating enzyme,either AADC or TD, would be in close proximity to any L-dopa produced bythe TMO, by virtue of both enzyme being encapsulated in the same matrix,conversion to dopamine readily proceed. Because tyrosine is less toxicthan L-dopa, it may be desirable to use a matrix to effect twoconversions, instead of just one. The present invention contemplatesencapsulating more than one reaction center per matrix. Yamanka et al.J. Sol-Gel Sci. & Tech. 7:117-21 (1996).

In another embodiment, the enzyme monophenol monooxygenase (MMO E.C.1.14.18.1), or tyosinase, is encapsulated as the reaction center. MMO isthe key enzyme in melanin synthesis, catalyzing the first two steps ofthe pathway: dihydroxylation of L-tyrosine to L-dopa and oxidation ofL-dopa to dopaquinone. The former reaction is termed cresolase activity,and the later reaction is termed catecholase activity. A number ofassays have been used to measure the tyrosinase hydroxylase and dopaoxidase activities, including spectrophotometric, radiometric, HPLC andelectrometric methods. Winder, J. Biochem. Biophys. Methods 28:173-83(1994); Vachtenheum et al., Analytical Biochem. 146:405-10 (1985). MMOsfrom a number of different sources are known. Kenji Adachi et al.Biochem. Biophys. Res. Comm. 26 (1967); Seymour H. Pomerantz,Tyrosinases (Hampster Melanoma) 620-626; Duckworth et al., J. Biol.Chem. 245:1613-25 (1970); Steiner et al., Analytical Biochem, 238:72-75(1996). Oxidation of o-diphenols to benzoquinones is referred to ascatecholase activity. Although catecholase activity of MMO may reducethe production of the desired therapeutic L-dopa product, engineering ofthe sol-gel matrix may allow for increased production of the diphenolproduct. For example, it has been reported that administration ofliposome-entrapped tyrosinase to rat increases levels of L-dopa in ratplasma. Miranda et al. Gen. Pharmacol. 24:1319-22 (1993). In the presentinvention, if the matrix is administered in the brain, then the increasein L-dopa would occur where it would have the greatest therapeuticeffect.

In another aspect of the present invention, modulation of dopamrine andrelated neurotransmitters may have use in treatment for cocaineaddiction. See U.S. Pat. No. 5,189,064. Chronic cocaine users mayexperience dopamine deficiency, and dopamine supplementation like thatcontemplated by the present invention may reduce the feeling ofdysphoria inadequate stimulation attributable to depressed dopaminelevels, which invites readministration of the drug or recividism.

In another aspect, the present invention contemplates applying thematrix-based technology to modulate the availability of any compounds byaugmenting the enzymes found in the biological pathway for any suchcompounds. For example, tryptophan is converted into5-hydroxy-tryptophan by the enzyme tryptophan hydroxylase withconcomitant conversion of tertrahydrobiopterin to dihydrobiopterin. AADCthen converts 5-hydroxy-tryptophan to serotonin, which is aneurotransmitter. Serotonin is found in the gastrointestinal tract andin the brain, where it is synthesized locally in the pineal gland.Monoamine oxidase converts serotonin into 5-hydroxy-indoleacetaldehyde,which aldehyde dehydrogeanse metabolizes into 5-hydroxy-indoleaceticacid. The present invention contemplate encapsulation any one or more ofthe enzymes in the serotonin pathway either to produce, as was discussedfor the dopamine example discussed above, or to degrade serotonin invivo as clinically necessary to treat any disease or condition. Thus,those of skill in the art may be able to use the present invention tomodulate any biological pathway using enzymes of such pathway asreaction centers.

Some other possible enzymes, which may be useful in neuropharmacologyand which may be used as reaction centers in the present invention, andthe reaction that they catalyze, are listed below: Enzyme Chief ReactantChief Product Choline acetyl CoA + CoA + o-acetylcholineacetyltransferase choline Phenylalanine 4- L-phenylalanine L-tyrosinemonooxygenase Dopamine dopamine norepinephrine β-monooxygenase(noradenaline) Noradrenalin N- noradrenalin adrenaline methyltransferase(epinephrine) Monoamine oxidase norepinephrine 3,4-dihydroxy-phenylglycolaldehyde Catecholamine-O- norepinephrine 3-O- methyltransferase methylnorepinephrine (COMT) Histidine histidine histaminedecarboxylase Histamine histamine 1-methylhistamine methyltransferaseDiamine oxidase histamine 5-imidazole acetic acid Diamine oxidase1-methylhistamine 1-methylimidazole acetic acid L-Glutamic glutamateγ-aminobutyric acid-1- acid (GABA) decarboxylase GABA-α- γ-aminobutyricglutamate and oxoglutarate acid (GABA) (plus succinic transaminaseα-oxoglutarate) semialdehyde Serine hydroxy- L-serine glycine methylaseSee generally Enzymes (Dixon et al. eds; 3d ed. 1979).

In addition to these illustrative examples, the present inventioncontemplates providing any biologically active agents to treat a diseaseor condition. For example, in the nervous system, chronic, low-leveldelivery of trophic factors is sufficient to maintain the health ofgrowth-factor dependent cell populations. In chronic disorders such asAlzheimer's disease and Huntington's disease, long-term delivery of oneor more neurotrophic factors such as NGF, BDNF, NT-3, NT-4/5, CNTF, GDNFand CDF/LIF may be required to maintain neuronal viability. These growthfactors cannot be delivered through systemic administration as they areunable to traverse the blood-brain barrier. Therefore, it may benecessary to deliver such neurotrophic factors into the central nervoussystem as a prodrug that is able to cross the blood-brain barrier. Thepresent invention contemplates preparing prodrugs of such biologicallyactive agents, and using an encapsulated reaction center to convert theprodrug into such an agent in the central nervous system. For example,CNTF is a potential therapeutic agent for Huntington's disease. Emerichet al. Nature 386:395-99 (1997). As discussed in more detail below, oneof skill in the art could prepare a prodrug for CNTF so that a specificencapsulated reaction center would be capable of converting the prodrugto CNTF. As one example of such a matching between a prodrug and areaction center, insulin may be obtained from recombinant proinsulin byreaction with carboxypeptidase or trypsin. Markvicheva et al. App.Biochem. Biotechnol. 61:75-84 (1996).

In certain embodiments of the present invention, an antibody may beencapsulated as the reaction center and the matrix administered to thesubject. The matrix would be used to isolate deleterious biologicallyactive agents from the subject in the matrix, as opposed to modifying aswould a reaction center that reacts with such a biologically activeagent. After all the antibodies have bound their corresponding hapten,the matrix may or may not be removed from the subject.

In certain embodiments matrices of the present invention may be used asprodrug activators in vivo after administration of the matrix and, ifnecessary, any prodrug, to a subject. Alternatively, the presentinvention contemplates using a matrix to convert a prodrug into abiologically active agent ex vivo, whereupon the biologically activeagent is administered to a subject, as described in more detail in U.S.Pat. No. 5,378,232.

5.2.2. Enzyme Replacement, Augmentation, or Supplementation

In other aspects of the present invention, a reaction center isencapsulated in a matrix to replace or augment some lost biologicalactivity that is generally present in a healthy subject, e.g., arewithout the disease or condition to be treated. Certain embodiments ofthe production of dopamine described above are examples of suchreplacement or augmentation. The encapsulated reaction center mayrestore or augment vital metabolic functions, such as the removal oftoxins or harmful metabolites. Lost biological activity might resultfrom loss of activity generally, or loss of activity in some location,e.g., a specific type of tissue. The loss may be attributable to agenetic defect, for example, an inborn error of metabolism, or may becaused by some disease or condition. If loss of function is complete,then this aspect of the invention may be referred to as enzymereplacement, whereas if loss of function is only partial, then thisaspect of the invention may be referred to as enzyme augmentation. Inother embodiments, although there has been no loss of enzyme function,enzyme augmentation may produce a desirable therapeutic effect. In otherembodiments, the enzyme encapsulated is new to the subject, that is,there is supplementation. Through use of certain embodiments of thepresent invention, homeostasis of particular substances can be restoredand maintained for extended periods of time.

Based on conditions and diseases that those of skill in the art know toresult from loss of a particular enzymatic activity, reaction centersthat replace or augment lost or diminished biological activity may bereadily identified. Certain embodiments of the present invention haveadvantages over more conventional types of enzyme replacement therapy(ERT), which often rely on administration of an enzyme whose activity islost or diminished. The matrices of the present invention for the mostpart may be biocompatible and immunoisolatory with respect to anyencapsulated reaction center, whereas enzyme administration can resultin hypersensitivity and/or anaphylactic reaction during or immediatelyafter enzyme infusion. Brooks et al., Biochem. Biophys. Acta 1497:163-72(1998). The development of antibodies to any enzyme used in ERT maypreclude its use in long-term therapeutic regimes or following relapses.Likewise, the present invention may avoid the need to dermitize anenzyme used in ERT with polyethylene glycol (PEG). Goldberg et al.Biomedical Polymers 441-52 (Academic Press 1980). Certain embodiments ofthe present invention, by encapsulating an enzyme as the reactioncenter, prevent degradation, and thereby may provide for prolongedtreatment upon administration of the matrix. In contrast, for ERT,multiple infusions of the enzyme may be required for sustained therapy.Even erythrocyte-entrapped enzymes may show only modest increases inactivity. See, for example, Thorpe et al. Pediatr. Res. 9:918-23 (1975).

By way of illustration, ERT has been used to treat Gaucher's disease.See generally Morales, Ann. Pharmacother. 30:381-88 (1996). Gaucher'sdisease is caused by a genetic deficiency of the enzymeglucocerebrosidase, and results in accumulation of glucocerebrosidasewithin the reticuloedothelial system. Symptoms includehepatosplenomegaly, bone marrow suppression, and bone lesions. There arethree subtypes, of which the most common, type 1, is non-neuronopathic.Types 2 and 3, which are neuronopathic, may result in nerve celldestruction. Enzyme treatment first began with a placentally derivedform of glucocerebrosidase, Dale et al. Proc. Natl. Acad. Sci. USA73:4672-74 (1976), which has been replaced with a recombinant version ofthe enzyme. The enzyme treatment must be repeated every two weeks, andit has been effective in reducing hepatosplenomegaly, improving anemiaand thrombocytopenia, and general health. Numerous studies have beencompleted on ERT for Gaucher's disease. Magnaldi et al., Eur. Radiol.7:486-91 (1997); Ueda et al., Acta Paediatr. Jpn. 38:260-04 (1996);Lorberboym et al., J. Nucl. Med. 38:890-95 (1997); Charrow et al., ArchInter. Med. 158:1754-60 (1998). The present invention contemplatesencapsulation of this enzyme in a matrix and administration to a subjectsuffering from Gaucher's disease for treatment.

ERT has also been effected by using materials that act by slow release.For example, L-asparaginase has been loaded into nanoparticles composedof polymers that release the enzyme over a few weeks, or loaded intoerythrocytes, Updike et al. J. Lab. Clin. Med. 101:679-91 (1983). Theenzyme may be useful for treating cancer, especially acute lymphocyticleukemia. By encapsulating this enzyme as the reaction center in amatrix, the enzymatic activity may be present for longer periods thanthat made possible by using slow-release methods.

In another embodiment of the present invention, transport proteins suchas hemoglobin may be encapsulated to produce an artificial red bloodcell. In such an embodiment, the matrix must be of appropriatemorphology to travel throughout the vasculature of a subject.

In addition to the examples already discussed, almost any naturallyoccurring enzyme may be used in to augment or replace enzymaticactivity, and is therefore a candidate for this use. Some other possibleenzymes that may be used as reaction centers, and the disease orcondition that they may treat, are listed below. (These enzymes may beused to treat other diseases and conditions as well.) Disease or Enzymecondition Reference α-1,4-Glucosidase Type II glycogenosis (Pompe'sdisease) α-Galactosidase Fabry's disease (heart and kidney failure dueto ceramide accumu- lation) α-L-iduronidase mucopolysaccha- Kakkis etal. ridosis type I. Biochem. Mol. Med. 58: 156-67 (1996) β-glucuronidasemucopolysaccha- O'Connor et al. ridosis type VII J. Clin. Invest. 101:1394-400 (1998) Aminolaevulinate Lead poisoning Bustos et al.dehydratase Drug Des. Deliv. 5: 125-31 (1989) Bilirubin oxidase jaundiceCatalase Acatalasemia Fibrinolysin Thromboembolic occlusive vasculardisease Glutaminase (e.g., Cancer from Pseudomonas putrefaciens)Hemoglobin Respiratory Heparinase (e.g., Extracorporeal from Flavobacte-circulation rium heparinum) L-arginine urea- Hyperargininemia Wissmannet al. hydrolase Somot. Cell Mol. (A1), Arginase Genet. 22: 489-98(1996) Liver microsomal Liver failure Brunner et al. enzymes (e.g.,Artif. Organs from rabbit liver) 3: 27-30 (1979); U.S. Pat. No.5,849,588 Phenylalanine Phenylketonuria Bourget et al. ammonia lyaseBiochim Biophys (e.g., from Acta 883: 432-48 Rhodotorula (1986)glutinis) Streptokinase (e.g., Thromboembolic from Streptococcus sp.)occlusive vascular disease Superoxide dismutase Inflammatory LedwozywActa (e.g., from bovine diseases thought Vet Hung 39: 215-24 liver),catalase to be mediated (1991); Turrnes et by oxygen free al. J. Clin.Invest. radicals, e.g., 73: 87-95 (1984) bleomycin-induced lung fibrosisTerrilythin Peritonitis Tyrosinase Liver failure UDP GlucuronylJaundice, liver transferase (e.g., disease from rabbit liver) Urea cycleLiver failure enzymes Urease Renal failure Uricase (e.g., Hyperuricemiafrom hog liver) due to gout Urokinase (e.g., Thromboembolic from humanurine) occlusive vascular diseaseSee generally Klein et al. TIBTECH July, 1986, 179-86.

5.2.3. Addiction Neutralization

In another aspect of the invention, the encapsulated reaction center ischosen to degrade biologically active agents that may result inaddiction. Efforts to combat addiction, e.g., cocaine addiction, haveincluded inducing anti-drug antibodies specific to the drug. Seegenerally U.S. Pat. No. 5,840,307. In certain embodiments, the presentinvention may help to neutralize the addiction, or in other words, causeaddiction neutralization. For example, investigators have encapsulatedalcohol dehydrogenase and/or acetaldehyde dehydrogeanse in humanerthrocytes and reported the continuous degradation of ethanol for up toseventy hours. Lizano et al. Biochem. Biophys. Acta 1425:328-36 (1998).See also U.S. Pat. No. 5,759,539. Encapsulation of either or both ofthese enzymes in a matrix may allow for the complete metabolization ofethanol upon administration of the matrix, which would thereby combataddiction by neutralizing the addictive agent, ethanol.

In another example, catalytic antibodies have been elicited that arecapable of aiding hydrolysis of the cocaine molecule. Landry et al.Science 259:1899-1901 (1993). See also U.S. Pat. No. 5,730,985.Encapsulation of such catalytic antibodies in a matrix of the presentinvention and administration to a subject addicted to cocain may allowfor neutralization of any ingested cocaine. The present invention, byencapsulating the catalytic antibody, may avoid some of the drawbacksusually associated with passive antibody therapy. In the same fashion,reaction centers targeted at biologically active agents responsible forother types of addition are known to those of skill in the art, and maybe used in a similar fashion.

5.2.4. Mutagenic Assays

In another aspect of the invention, the present invention contemplatesusing matrices as metabolic activating systems for use, for example, astoxicology screens for cytotoxic and pharmaceutical compounds in vivo.Such a use may reduce the need for laboratory animals for toxicologytesting. Numerous efforts have been made to prepare human liverepithelian cell lines, and liver cell and tissue culture systems forsuch uses. See, for example, U.S. Pat. Nos. 5,849,588 and 5,759,765. Inone report, enzymes responsible for deactivation of many endogenoustoxins have been isolated and covalently bound onto a hemocompatibleform of agarose support. Brunner et al., Artif. Organs 3:27-30 (1979).In a like manner, the present invention contemplates encapsulating suchenzymes in matrices of the present invention and evaluating suspectedmutagens. See generally Rueff et al Mutat. Res. 353:151-76 (1996).Enzymes usually found in the liver, such as cytochrome P-450 forexample, may be used in this or related embodiments. Janig et al. ActaBiol. Med. Ger. 38:409-22 (1979).

5.2.5. Tissue Assist Devices

In addition to the many methods and uses described herein in which thesubject matrices are administered for in vivo use, the matrices of thepresent invention may be used ex vivo. Many of the teachings hereindescribed for in vivo use apply as well to ex vivo use (and visa-versa).

In one aspect of the present invention, one example of an ex vivo use isa tissue assist device, and in certain embodiments, an organ assistdevice. In such a device, matrices encapsulating one or more reactioncenters could be used to replace, augment or supplement the biologicalfunction of an organ or other tissue. It is important to note that forthis embodiment (and others described herein in which the prodrugconverted by the reaction center is potentially deleterious to thesubject being treated), it is not always necessary that the prodrug beconverted into the same agent(s) that the enzymes and other catalystsusually present in the tissue would have were it to react with theprodrug. In certain embodiments of the subject invention, it is not theproduct of the prodrug that is critical; instead it is thetransformation of the prodrug into a less toxic or otherwise undesirablecompound(s) that is the primary concern. For example, this principleapplies to certain of the embodiments used for addition neutralizationdescribed above as well as certain tissue assist devices.

One example of such an assist device is particularly well-suited to thesubject matrices is an hepatic assist device. Liver transplantation hasbecome widely accepted as an effective treatment for chronic and acuteliver disease. One of the major problems associated with thetransplantation process, however, as been the need for an effectivemeans for providing temporary support for patients awaiting an availabledonor organ. Extracorporeal devices that are effective fofliver supporthas proven more elusive. See, for example, Takahashi et al., DigestiveDiseases and Sciences 36(9) (1991).

In certain embodiments of the present invention, one or more enzymesgenerally localized in the liver of a patient could be encapsulated in asubject matrix, and blood and other bodily fluids of the patient couldbe passed through and over these matrices ex vivo to augment thebiological activity usually associated with the liver. Functions of theliver that could be addressed by such liver assist devices include,among others, carbohydrate, fat and protein metabolism anddetoxification of drugs, hormones and other substances. At the pointthat the encapsulated reaction centers of the matrices are exhausted orotherwise less efficient than desired, they may be readily replaced byproviding with new and more efficient matrices.

A variety of reaction centers could be encapsulated for such a liverassist device. Examples include cytochrome P-450, other enzymes usuallylocated in the liver, a less than highly purified mixture of biologicalsisolated from livers, transformed cells such as those derived fromhepatoblastoma cell lines (Sussman et al., Hepatology 16:60-65 (1992)),cultured or isolated hepatocytes (U.S. Pat. No. 5,866,420; Rozga et al.,Hepatology 17:258-65 (1993); Rozga et al., Ann. Surg. 217:502-11(1994)), cells from hepatocarcinoma-derived cell lines (Richardson etal., J. Cell Biol. 40:236-47 (1969); Aden et al., Nature (London)282:615-16 (1970)), Kupffer cells and other biologicals that are capableof replacing, augmenting or supplementing the biological function of theliver. For other examples of possible biologicals and liver assistdevices, see, for example, U.S. Pat. Nos. 6,008,049, 5,849,588,5,290,684, 5,270,192, 5,043,260, 4,853,324, 3,734,851; and WO 93/16171.Sources of suitable enzymes and biologicals for a livers assist devicefor humans include, for example, porcine and other mammals. Inparticular, use of certain biologicals, including for examplehepatocytes, has proved difficult because their instability, andencapsulation of such biologicals in matrices of the present inventionmay improve their stability.

A variety of bioreactor techniques known to those of skill in the arecould be used with such an assist device, including for example, hollowfiber techniques, static maintenance reactor systems, fluidized bedreactors, microporous membranes and flat-bed, single-pass perfusionsystems. See, for example, U.S. Pat. Nos. 4,200,689, 5,081,035,3,997,396; and WO 90/13639; Halberstadt et al., Biotechnology andBioengineering 43:740-46 (1994).

In addition to liver assist devices, other organs or functions of apatient could be treated using matrices of the present invention exvivo.

5.3. Matrices

The concept of encapsulating or immobilizing a reaction center in or ona matrix of some kind is well precedented. For example, significantefforts have been made to immobilize enzymes on solid supports. Handbookof Enzyme Biotechnology (2d ed., ed. Wiseman 1985). In these and otherexamples, encapsulation or immobilization of the reaction center mayimpart desirable characteristics on the reaction center.

A number of matrix chemistries that may be used in the present inventionhave been used to immobilize enzymes or biologically active agents. Forinstance, cells have been attached to glass beads and implanted in rats.Cherksey et al. Neuroscience 75:657-64 (1996). Reaction centers may beimmobilized on a type of porous zirconia. Huckel et al., J. Biochem.Biophys Methods 31:165-79 (1996). Alternatively, reaction centers may beattached to supports through silane coupling. Weetall, Appl. Biochem.Biotechnol. 41:157-88 (1993). Biologics may be immobilized within acomposite fibre by using a gel formation of cellulose derivative andmetal alkoxide, e.g., titanium isopropoxide. Hatayama et al. J. Sol-GelSci. & Tech. 7:13-17 (1996); Ohmori et al. J. Biotechnol. 33:205-09(1994). Poly(vinyl alcohol) synthetic polymer foams may be used. Li etal. J. Biomater. Sci. Polm. Ed. 9:239-58 (1998). Other polymers known inthe art may be used. See, for example, U.S. Pat. Nos. 5,529,914 and5,780,260; WO 93/16687. As described in greater detail below,inorganic-based or silica-based sol-gel matrices are contemplated by thepresent invention. Some examples of suitable inorganic-based matricesinclude those disclosed in the following references: Mazei et al., J.Materials Chemistry 8:2095-101 (1998); Yoldas, J. Mater. Sci. 1098-92(1986); and Curran et al., Chemistry of Materials, 10:3156-66 (1998).

One feature of the matrix in certain embodiments of the presentinvention is its ability to prevent leaching of any encapsulatedreaction center, at least to the extent necessary for the intended useof the matrix. In certain embodiments of the present invention, theremay be negligible leaching. In others, there may be some leaching, butusually only a small amount over time. If leeching proses to beexcessive, either the material to be encapsulated, e.g., a reactioncenter or an additive, or the sol-gel matrix may be modified to improveleaching characteristics, e.g., reduce leaching. For example, to reduceleaching, the reaction center may be derivatized to increase its size.For example, an enzyme may be chemically modified to create derivativesby forming covalent or aggregate conjugates with other chemicalmoieties, such as glycosyl groups, lipids, phosphate, acetyl groups,PEG, and the like. Covalent derivatives may be prepared by linking thechemical moieties to functional groups on amino acid sidechains of theprotein or at the N-terminus or at the C-terminus of the polypeptide.Alternatively, a fusion or chimeric polypeptide retaining at least someof the activity of the enzyme may be used. Alternatively, the reactioncenter of additive may be attached to the sol-gel matrix in somefashion, e.g., by covalently bonding.

The manner in which a reaction center is encapsulated in a matrix, be itfor example by physical entrapment, covalent attachment, or some otherphysical attraction, may affect the properties of such reaction center.For example, the micro environment around any covalently attachedreaction center may differ from that encountered by the same reactioncenter encapsulated during gelation of the sol-gel, and any differencemay affect the activity of the center. Thus, the present inventioncontemplates adjusting encapsulation, if necessary, for each intendeduse.

In preparing any matrix, the encapsulated material, e.g. the reactioncenter and additives, must be robust enough to retain their usefulnessafter being encapsulated. For example, many biological materials may notbe able to survive the high temperatures and harsh conditions requiredto prepare some inorganic materials. Consequently, such inorganicmaterials may not be used with sensitive biolgicals. In the presentinvention, matrices are matched with the reaction center(s) oradditive(s) to be encapsulated therein so as to retain sufficientactivity of the reaction center.

Another feature of the present invention is the ability of the matrix tostabilize, in certain cases, the encapsulated reaction center. Forexample, the present invention may protect against degradation of anyencapsulated biological material by naturally occurring systems, such asproteases. The matrix may protect against thermal denaturation of anyencapsulated biological materials. Finally, the matrix may even assistin the correct re-folding of any denature polypeptide chain.Heichal-Segal et al. Bio/Technology 13:798 (1995).

5.3.1. Silica-Based Sol-Gel Matrices

In one aspect of the present invention, reactions centers areencapsulated in silica-based sol-gel matrices. Silica-based sol-gelshave been applied to encapsulate a wide range of materials, includingbiological materials, small organic molecules, antibodies, antigens, andorganic catalysts. See, for example, Ellerby et al. Science 255:1113-15(1992); Dave et al. Analytical Chem. 66:1120-27 (1994); Avnir et al.Acc. Chem. Res., 28:328-34 (1995); Avnir et al. Chem. Mater., 6:1605-14(1994) (listing encapsulated purified enzymes and whole-cell extractsand whole cells); Biochemical Aspects of Sol-Gel Science and Technology(eds. Avnir et al. 1996); Shtelzer et al. Biotechnol. Appl. Biochem.15:227-35 (1992). Biological materials encapsulated in inorganic-basedor silica-based sol-gel matrices have retained significant activity fora substantial time period.

Inorganic-based sol-gels, and in particular, silica-based sol-gels, havea variety of characteristics that are useful for encapsulation ofreaction centers and implantation in vivo. See generally Dunn et al.Acta Mater. 46:737-41 (1998); Avnir et al. Chem. Mater., 6:1605-14(1994). Any or all of these features may or may not be present inparticular embodiments of the present invention. Some such featuresinclude: stability to heat, light (no photodegredation), and electricalcurrent (no electrochemical degradation); transparent in the visibleregion and into the UV-Vis region; controllable surface area andporosity (average pore size and pore size distribution); possibility ofcontrolling conductivity by appropriate use of other inorganic alkoxidesduring preparation of the gel or addition of additives; capable of beingreadily modified chemically; improved stability of any encapsulatedmaterial, e.g., reaction center or additive, because of the rigidmatrix; little or no leaching of any encapsulated material; readilymanipulated in a variety of physical morphologies; and isolatory of anyencapsulated material from the surrounding environment, except for anysubstance that is able to diffuse into the matrix. Many of thesefeatures are explained in greater detail below.

One area of interest involves using doped sol-gels as chemical sensors.As part of that effort, sol-gels have been used to encapsulate enzymesand antibodies. Avnir, Acc. Chem. Res., 28: 328-334 (1995); Akbarian etal. J. Sol-Gel Sci & Tech. 8:1067-70 (1997). Imuunosensors have beenprepared using sol-gel technology. Wang et al. Anal. Chem. 15:1171-75(1998). For example, the enzyme glucose oxidase has been examined uponencapsulation in a silica-based sol-gel matrix for use as a glucosesensing material. Yamanaka et al. Chem. Mater. 4:495 (1992); Audebert etal. Chem Mater. 5:911-13 (1993). Such sol-gel preparations have beenused as electrodes for electrochemical assays of glucose concentrations.Sampath et al. J. Sol-Gel Sci. & Tech. 7:123-28 (1996).

Another area of interest of silica-based sol-gel technology has beenencapsulating enzymes for use as organic catalysts in a variety ofapplications, including synthesis of chiral materials. Jaeger et al.TIBTECH 16:3967-403 (1998).

Silica-based sol-gel matrices have also been used as controlled-releasecarriers of biologically active agents. See, for example, U.S. Pat. No.5,849,331 and WO 97/45367.

(a) Preparation

Modifications in well-known sol-gel processes permit the incorporationof enzymes or other biologically derived reaction centers insilica-based sol-gel matrices. See generally Avnir et al. Chem Mater.6:1605 (1994); U.S. Pat. Nos. 5,824,526, 5,650,311; 5,650,311;5,371,018; 5,308,495; 5,300,564; 5,292,801.

Silica-based sol-gel matrices of the present invention may be preparedin the sol-gel method by polymerization of a metal alkoxide precursor.See generally Bruce Dunn et al. Chem. Mater., 9:2280-91 (1997). Thepolymerization process is well documented and known to proceed by theformation of colloidal silica particles. A suspension of these particlesis termed a sol. The synthesis generally involves the use of metalalkoxides which may undergo hydrolysis and condensation polymerizationreactions. The preparation process can ordinarily be divided into thefollowing steps: forming a solution, gelation, ageing, drying, anddensification. In the preparation of a silica-based matrix, one startswith an appropriate alkoxide, for example, Si(OC₂H₅)₄, tetraethylorthosilicate or TEOS, or Si(OCH₃)₄, tetramethyl orthosilicate or TMOS,which is mixed with water and a solvent, e.g., the alcohol of thealkoxide, ethanol or methanol, to form a solution. A number of reactionsresult, including hydrolysis, which leads to the formation of silanolgroups Si—OH, and condensation, which gives siloxane Si—O—Si groups.

There are several parameters which influence the hydrolysis andcondensation polymerization reactions, including the temperature,solution pH, particular alkoxide precursor and solvent, and relativeconcentrations of the alkoxide precursor, water, and solvent. Suchparameters may be important to retaining activity when the reactioncenter encapsulated is an enzyme or other biological. For example, inencapsulating enzymes, greater enzyme activity may been preserved by notadding any alcohol at the start of polyermization. Another improvementmay result from buffering the reaction solution to some pH suitable forany pH-sensitive materials to be encapsulated after the acid-catalyzedhydrolysis of the oxysilanes. Ellerby et al. Science 255:1113-15 (1992);Rietti-Shati et al. J. Sol-Gel Sci. & Tech. 7:77-79 (1996).

Initial hydrolysis of the precursor alkoxide is catalyzed by protons orhydroxide ions. It is possible to control the matrix characteristics bycontrolling the rates of the individual steps by which the matrix iscondensed. Acidic catalysis tends to increase the rate of hydrolysis anddisfavors the condensation reactions necessary to form the sot-gel,whereas base hydrolysis produces rapid condensation. If the reactioncenter to be encapsulated is not sensitive to pH conditions, theformation of the gel matrix can be achieved fairly rapidly. However, inthe case of reaction centers or additives which may be sensitive toextreme pH conditions, such as enzymes, the pH of the sol must beadjusted prior to addition. Hence, preparation of the silica-basedsol-gel may involve buffering the sol before the addition of thereaction center or other additives. For example, to retain the activityof bacteriorhodopsin, the solution was buffered to pH 9 after additionof the polypeptide. Weetall et al. Biochem Biophys. Acta 1142:211-13(1993).

As the hydrolysis and condensation of polymerization reactions continue,viscosity increases until the solution ceases to flow. This sol-geltransition is irreversible, and at this stage the one-phase liquid istransformed to a two-phase system. The gel may consist of amorphousprimary particles of variable size (5-10 nm or smaller) with aninterstitial liquid phase. At this stage the pores have yet to shrinkand the liquid phase fills the pores. After gelation, gels are generallysubjected to an aging process during which the gels are sealed and verylittle solvent loss or shrinkage occurs. Condensation reactionscontinue, increasing the degree of cross-linking in the network.

The drying process involves the removal of the liquid phase. Ambienttemperature evaporation may be employed, and there is considerableweight loss and shrinkage. It is at this stage that pore collapse mayoccur, deceasing pore size and thus decreasing the solvent volume. Thecombination of these effects causes an increase in the interactionbetween the reaction center and the matrix. The final stage of thesol-gel process is that of densification. It is at this point that thegel-to-glass conversion occurs and the gel achieves the properties ofthe glass. Matrices are found to contract to one eighth the pre-driedvolume and are termed “xero-gels.” The drying process may affect theaccessibility of any encapsulated reaction center, and by adjusting suchprocess, the present invention contemplates another means of influencingthe activity of any encapsulated reaction center. Wamboldt et al. J.Sol-Gel Sci & Tech 7:53-57 (1996).

(b) Composition and Characteristics

Any number of alkoxide precursors may be used in preparing silica-basedsol-gel matrices of the present invention. Those silica-based sol-gelmatrices prepared from oxysilanes other than Si(OR¹)₄ are known asorganically modified silica matrices, or Ormosil matrices. In preparingthe matrices of the present invention, for example, alkoxides of theform Si(OR¹)₄, R²Si(OR¹)₃, R²²Si(OR¹)₂, R²³Si(OR¹) may be used, in whicheach R¹ is independently methyl, ethyl, or any lower-weight alkyl(although the identity of R¹ is usually the same in any type ofoxysilane), and R² is independently any alkyl, aryl, or othersubstituent that does not interfere substantially with formation of thesol-gel, as discussed in more detail below. A significant differencebetween R¹ and R² is that the R¹ alkoxide the majority of R¹ ishydrolyzed during gelation, whereas the R² substituent remains part ofthe matrix. Because R² is not hydrolized but remains in the sol-gelmatrix, the identity of R² may have a significant affect on the sol-gelmatrix and any material encapsulated therein. In contrast, for R¹, muchof which is hydrolized during gelation, may not constitute a significantpercentage of the sol-gel matrix that results. Even so, the identity ofR¹ may be important to the reaction, because HOR¹, which is producedupon hydrolysis of the oxysilane, may affect the formation of thesol-gel and any material encapsulated therein. Accordingly, for example,some biolgicals to be encapsulated may be stable to some HOR¹ and notothers. In certain preferred embodiments, the alkoxide used is Si(OR¹)₄,in which R¹ is methyl or ethyl.

In certain embodiments, R² may contain functional groups. For example,aminopropyl, which has an amine functional group, and mercaptopropyl,which has a thiol functional group, have been used as R² in preparingsol-gel matrices from R²Si(OR¹)₃ and mixtures of R²Si(OR¹)₃ andSi(OR¹)₄. Collino et al. J. Sol-Gel Sci. & Tech 7:81-85 (1996); Ventonet al. Biochim Biophys Acta 1250:117-25 (1995). Such sol-gel matriceswere used to prepare thin films. Almost any chemical moiety may be usedas R² in the present invention as long as any functional groupscontained therein are not adverse to formation of the sol-gel matrix.For those functional groups that may not be compatible with the sol-gelchemistry, it may be possible to protect them using standard protectiontechnologies know in the art of organic chemistry and then deprotectthem after preparation of the sol-gel matrix is complete.

Functional groups may be used to increase the stability or reactivity ofany encapsulated reaction center, especially when the reaction center isa biologic. For example, functional groups having hydrolizablefunctional groups, such a phenol or amine, may affect the local pH,thereby improving reactivity or stability of the encapsulated reactioncenter, or directly assist catalysis or stabilize an enzyme.Alternatively, functional groups may affect the characteristics of thesurface of the matrix, which may affect the biocompatability of thematrix. In addition, by incorporating functional groups into thematrices of the present invention, the exterior characteristics of thematrix may be altered by derivitization. For example, chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups, PEG, and thelike, could be attached to the surface of the matrix though suchfunctional groups.

In other embodiments of the present invention, R² may incorporatechemistry that allows for covalent attachment of any reaction center oradditive directly to the sol-gel matrix. For example, a reaction centeror additive could be covalently attached to the silicon of an oxysilaneas R² through a linker bound to the silicon or other morganic. Such amodified silica alkoxide could be reacted with nonsubstituted silicaalkoxides to form the sol-gel of interest. In another embodiment, themoiety covalently attached to the silica alkoxide could be a biotingroup, and the reaction center or additive could be attached to avidin.The biotin/avidin interaction would effectively attach the reactioncenter or additive to the silica oxide framework of any sol-gel matrix.An antibody/hapten pair could be used in the same fashion.

For certain embodiments, the alkoxide used may be a single silicaalkoxide, or a mixture of silica alkoxides. Reetz et al. J. Sol-Gel Sci.& Tech. 7:35-42 (1996). When using silica oxides of structureR²Si(OR¹)₃, R²²Si(OR¹)₂, R²³Si(OR¹), it may be necessary to react themwith sufficient Si(OR¹)₄ to allow for adequate gelation and formation ofa physically robust matrix. For instance, the oxysilanes substitutedwith non-hydrolizable substituents may constitute 0.1, 1, 2, 5, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the oxysilane usedin the preparation. If a oxysilane has non-hydrolizable substituents,the percent of that oxysilane may need to be less to ensure sufficientgelation.

Modification of the framework of a silica-based sol-gel by variation ofthe precursor alkoxide presents the possibility of tailoring themicroenvironment of the encapsulated reaction center. In this fashion,it is possible to, for example, maximize the reactivity of anyencapsulated reaction center. Increasing the number of alkyl groups aswell as increasing chain length of the alkyl groups in the precursormaterial may produce increased matrix hydrophobicity. Suchhydrophobicity, for example, may be conducive to stabilization of thereaction center. The local pH within the sol-gel may also be affected bythe chemical identity of the matrix. In one report, the activity of anencapsulated enzyme increased upon using a mixture of oxysilanes withvarious alkyl substituents. Reetz et al. Angew. Chem. Int. Ed. Engl.34:301-303 (1995). By varying the chemical identity and ratio ofdifferent oxysilanes, the present invention contemplates customizing thesol-gel matrix for each encapsulated reaction center, e.g., to maximizecatalytic activity. Other features of the matrix may depend on thechemical identity of the oxysilane precursors, for example wettability,which may affect the biocompatibility of any matrix upon implantation.For example, cell adhesion and growth may depend on the wettability ofthe matrix. Altankov et al. J. Biomed. Mater. Res. 30:385-91 (1996);Altankov et al. J. Biomater. Sci. Polym. Ed. 8:299-310 (1996).

Pore size is an important characteristic of any sol-gel matrix, becauseit may affect what materials, e.g., prodrugs, may diffuse in and out ofthe matrix, and the leachability of any encapsulated reaction center(s)and/or additive(s). A number of reports indicate that the pore sizeand/or shape may be varied by adjusting the synthetic conditions bywhich any material is encapsulated in a sol-gel. Dave et al. ACS Symp.Ser. 622:351 (1996). The present invention contemplates pore sizesranging from the angstrom level to the micron level.

In addition to preparing silica-based sol-gel matrices from silicaoxides, other oxides, including metal oxides, may be used to encapsulatea reaction center in inorganic-based sol-gel matrices. In one report,glucose oxidase was encapsulated within vanadium pentaoxide, and theresulting sol-gel was used in electrochemical studies. Glezer et al. J.Am. Chem. Soc., 115:2533-34 (1993). A vanadium alkoxide has beenco-condensed with TEOS, thereby imparting the properties, includingreactivity, of oxovanadium(V) functional groups to the matrix. Stiegmanet al. Chem. Mater. 5:1591-94 (1993). Oxysilines and other metal oxidesmay be combined in any sol-gel matrix. Silica-based sol-gel matrices inwhich redox active metal ions constitute part of the sol-gel frameworkmay prove useful in promoting reactions involving electron transferssuch as reductions and oxidations within the sol-gel itself. Forexample, an electron source foreign to the matrix may transfer anelectron to a reaction center encapsulated in such a mixed metal sol-gelby electron transfer through a pathway involving the metal atoms in theframework of the sol-gel. Soghomonian et al. Chem. Mater. 5:1595-97(1993).

The local environment, or micro-environment, immediately surrounding anyencapsulated reaction center of the present invention may play animportant role in affecting the capability of such reaction center tocatalyze the conversion of prodrug to biologically active agent. Anumber of studies have been completed to better characterize the natureof the microenvironment around any encapsulated material in a sol-gel.Samuel et al. Chem Mater., 6:1457-61 (1994); Zheng et al. Anal Chem.69:3940-49 (1997); Dave et al. J. Sol-Gel Sci & Tech. 8:629-34 (1997);Avnir et al. J. Phys. Chem. 88:5956-59 (1984). By adjusting any of thevariables delimited above, the properties of the silica-based sol-gelmatrix may be readily tailored by one of skill in the art to thereaction center encapsulated.

5.3.2. Other Features of the Matrix

The matrix may be immunoisolatory with respect to the reaction center orother contents. Use of immunoisolatory matrices allows the implantationof alkogenetic or xenogeneic reaction centers and other additives,without a concomitant need to immunosuppress the subject. Usingimmunoisolatory matrices, it is possible to implant reactions centersthat are foreign to the subject, such as nonmamallian enzymes, providedthat critical substances necessary to the mediation of immunologicalattack are excluded from the implant. These substances may comprise thecomplement attack complex component Clq, or they may comprise phagocyticor cytotoxic cells; the instant immunoisolatory matrix protects againstthese harmful substances.

The present invention allows for coating or otherwise modifying theexterior of the matrix. Such a coating may render the matriximmunoisolatory. U.S. Pat. No. 5,676,943. For example, the coating orother modification may confer protection of the reaction center or othercontents from the immune system of the host in whom the matrix isimplanted, by providing a physical barrier sufficient to preventdetrimental immunological contact between the reaction center and otheradditives and the host's immune system. The thickness of a coating mayvary, but it will always be sufficiently thick to prevent direct contactbetween the reaction center and the elements of the host's immunesystem. The thickness generally ranges between 5 and 200 microns;thicknesses of 10 to 100 microns are preferred, and thickness of 20 to75 microns are particularly preferred. Types of immunological attackwhich can be prevented or minimized by the use of a coating or othermodification include attack by macrophages, neutrophils, cellular immuneresponses (e.g. natural killer cells and antibody-dependent Tcell-mediated cytoloysis (ADCC), and humoral response (e.g.,antibody-dependent complement mediated cytolysis).

Various polymers and polymer blends can be used to manufacture acoating, including polyacrylates (including acrylic copolymers),polyvinylidenes, polyvinyl chloride copolymers, polyurethanes,polystyrenes, polyamides, cellulose acetates, cellulose nitrates,polysulfones, polyphosphazenes, polyacrylonitriles,poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymersand mixtures thereof.

The solvents used in conjunction with the above-identified polymers informing the coating will depend upon the particular polymer chosen.Suitable solvents include a wide variety of organic solvents such asalcohols and ketones generally as well as dimethylsulfoxide (DMSO),dimethylacetamide (DMA), and dimethylformamide (DMF) and blends of thesesolvents as well.

The coating may also include a hydrophobic matrix such as an ethylenevinyl acetate copolymer, or a hydrophilic matrix such as a hydrogel. Thecoating may be post-production coated or treated with an impermeableouter layer such as a polyurethane, ethylene vinyl acetate, silicon, oralginate.

The polymeric solution for the coating may also include variousadditives such as surfactants to enhance the formation of porouschannels and antioxidants to sequester oxides that are formed during thecoagulation process. Exemplary surfactants include Triton-X 100available from Sigma Chemical Corp. and Pluronics P65, P32, and P18.Exemplary anti-oxidants include vitamin C (ascorbic acid) and vitamin E.

In addition, anti-inflammatory agents can also be incorporated into thecoating to reduce immune response. Exemplary anti-inflammatory agentsinclude corticoids such as cortisone and ACTH, dexamethasone, cortisol,interleukin-1 and its receptors and agonists, and antibodies to TGF,interleukin-1, or gamma-interferon. Alternatively, these materials maybe added to the implant after formation by a post-coating or sprayingprocess. For example, the implant could be immersed in a solutioncontaining an anti-inflammatory agent.

Post-coating procedures can also be used to provide a protective barrieragainst immunogens and the like. For example, after formation, thematrix may be coated (e.g., by immersion, spraying or applying a flowingfluid, if applicable) with a surface protecting material such aspolyethylene oxide or polypropylene oxide to inhibit proteininteractions of the reaction centers of the matrix with entities of thesubject. Other protective coatings include silicon and hydrogels such asalginates. Derivatives of these coating materials, such as polyethyleneoxide-polydimethyl siloxane, may also be used.

The coating may be formed freely around the core without chemicalbonding, or alternatively, the coating may be directly cross-linked tothe material of the implant.

The present invention also allows for enclosing the matrix in amembrane. The membrane may allow for passage of substances up to apredetermined size but prevents the passage of larger substances. Morespecifically, the surrounding or peripheral region is produced in such amanner that it has pores or voids of a predetermined range of size. As aresult, the vehicle is selectively permeable. The molecular weightcutoff(MWCO) selected for a particular coating will be determined inpart by the use contemplated. Membranes useful in the instant inventionare ultrafiltration and microfiltration membranes.

If necessary, the present invention also allows for modification oradditions to be made to the matrix to support or strengthen the matrix.U.S. Pat. No. 5,786,216. For example, structural materials, such as ahollow tube or cylindrica support, may be encapsulated in the matrix toimprove its compression strength, tensile strength, or other properties.

The present invention also allows for attachment of a tether to thematrix to make implantation, placement, or recovery of the matrix morefacile. The present allow also contemplates altering the surface of thematrix, for example by encapsulating a structure that affects thesurface of the matrix, as an aid in fixing the matrix upon implantation.

5.3.3. Additives

Additives may be encapsulated in the matrix in addition to any reactioncenter or centers. Such additives may be used to alter the properties ofthe matrix. Investigations show that the addition of low concentrationsof organic molecules to sol-gels has very little effect on the networkformation of the sol-gel. Dunn et al. Chem. Mater 9:2280-91 (1997).

For example, additives that may be used in the present invention includesodium fluoride and polyethylene glycol. The use of polyethylene glycolin place of any alcohol during condensation of the sol-gel matrix mayimprove enzymatic activity of the encapsulated enzyme. Likewise, sodiumfluoride may be used and may improve enzymatic activity of theencapsulated enzyme. Avnir et al. Chem. Mater. 6:1605-14 (1994).

For example, additives may alter the porosity of the matrix.Alternatively, additives may be used to provide necessary reactants forany encapsulated reaction center. For example, coenzymes for a reactioncenter may be encapsulated along with the enzyme itself. For AADC andTD, a required cofactor is pyridoxal phosphate. Pyridoxal phosphate maybe encapsulated directly during preparation of the sol-gel matrix.Alternatively, pyridoxal phosphate itself may be prepared in a slowrelease formulation, and the formulation encapsulated. See generallyU.S. Pat. No. 5,759,582.

Alternatively, additives may improve the physical characteristics of thematrix, for example, for purposes of handling and administration of thematerial. As with the reaction center, additives may be physicallyencapsulated during the synthesis of the sol-gel, or they may becovalently attached to the matrix directly.

Another possible additive of the present invention allows for readydetection of the matrix after administration. In this fashion, thematrices of the present invention may also be used for diagnosticpurposes. Thus an X-ray contrast agent, such as a poly-iodo aromaticcompound, may be encapsulated in the matrix. Matrices according to theinvention may also contain paramagnetic, superparamagnetic orferromagnetic substances which are of use in magnetic resonance imaging(MRI) diagnostics. Thus, submicron particles of iron or a magnetic ironoxide may be encapsulated into the matrix to provide ferromagnetic orsupel) aramagnetic particles. Alternatively, paramagnetic MRI contrastagents, which principally comprise paramagnetic metal ions, such asgadolinium ions, ligated by a chelating agent which prevents theirrelease (and thus substantially eliminates their toxicity), may beencapsulated. Matrices of the present invention may also containultrasound contrast agents such as heavy materials, e.g. barium sulphateor iodinated compounds such as the X-ray contrast agents referred toabove, to provide ultrasound contrast media.

5.3.4. Matrix Morphology.

The matrices of the present invention may take any variety ofmorphologies. As a practical matter, the form of the matrix mayinitially be determined by the vessel in which the matrix issynthesized. Such matrices may subsequently be processed in order toproduce matrices of desired morphology. The size of any matrix maydepend on its intended use, and the present invention contemplatespreparing matrices having dimensions of centimeters (1, 10, 100, 1000cm), millimeters (1, 10, 100 mm), micrometers (1, 10, 100), nanometers(1, 10, 100), and picometers (0.01, 0.01, 1, 10, 100 pm). Alternatively,the size of a matrix may be referred to by mass, which the presentinvention contemplates may range anywhere from 1000 gm to 1 ng.

The matrix may be any configuration appropriate for providing sufficientactivity of the encapsulated reaction center necessary for its intendeduse. Possible morphologies include cylindrical, rectangular,disk-shaped, patch-shaped, ovoid, stellate, or spherical. For use in asubject, a matrix of the present invention may provide, in at least onedimension, sufficiently close proximity of any reaction centers to thesurrounding tissues of the subject, including the subject's bloodstream,in order to make any biologically active agent produced by the reactioncenter bioavailable.

If the matrix is to be retrieved after it is implanted, configurationswhich tend to prevent migration of the matrix from the site ofimplantation may be desirable; in contrast, if the matrix is intended tomigrate throughout a patient, other morphologies, such as sphericalcapsules small enough to travel in the recipient's blood vessels, may bedesirable. The degree of miniaturization of any matrix may affectmobility and localization of a matrix in a subject. Certain shapes, suchas rectangles, disks, or cylinders may offer greater structuralintegrity.

The surface area of the matrix may be important for its use. A greatersurface area may result in a greater observed activity with respect toany particular load of an encapsulated reaction center. In particular,small bead or sphere shaped materials ranging in size of radius from maybe desirable because they have increased surface area as compared toother morphologies. In order to increase further the surface area of amatrix, a powder of the matrix may be desirable. It may be desirable toenclose a matrix, especially if the matrix is in powder form, in acapsule. See, for example, U.S. Pat. Nos. 5,653,975; 5,773,286.

It may be possible to control the size of any matrix by using theaqueous core of reverse cellular micellar droplets as host reactors forpreparation of the matrix, as reported by Jain et al. J. Am. Chem. Soc.120:11092-95 (1998) for a silica-based sol-gel matrix. The matrixparticles prepared in such a fashion may be highly monodispersed andhave a narrow size distribution. Other hollow spheres may be used toprepare matrices of similar dimensions. See for example Caruso et al.Science 282:1111-13 (1998), U.S. Pat. No. 5,770,416, Lu et al., Nature398:223-26 (1999).

5.4. Reaction centers

A wide variety of compounds or materials may be used as the reactioncenter in the present invention. In general, any compound or materialthat converts a compound into a biologically active agent may beencapsulated. Alternatively, in other embodiments, any compound ormaterial that degrades a biologically active agent may be encapsulated.Alternatively, any compound or material exhibiting reactivity ofinterest may be encapsulated. Many possible reaction centers havealready been described in setting forth some of the uses of the presentinvention.

Possible types of reaction centers contemplated by the present inventioninclude, for example, enzymes, catalytic antibodies, antibodies, andnon-biologically derived catalysts. Many reaction centers may bebiologically derived. Numerous reports describe encapsulating enzymes insol-gels, and such teachings may be of assistance in embodiments of thepresent inventions. Zink et al. New J. Chem., 18:1109-15 (1994); Milleret al. J. Non-Crystalline Solids. 202:279-89 (1996); Ji et al. J. Am.Chem. Soc., 720: 222-23 (1998); Braun et al. J. Non-Castalline Solids147&148:739-43 (1992); Yamanaka et al. Chem. Mater. 4:497-500 (1992);Lin et al. J. Sol-Gel Sci. & Tech. 7:19-26 (1996). Catalytic antibodieshave been encapsulated in sol-gel matrices and the resulting matricesused in either a batch-wise operation or in a continuous flow apparatusfor preparative scale organic synthesis. Shabat et al. Chem. Mater.9:2258-60 (1997). Optically active polypeptides have been encapsulatedwith retention of activity. For example, bacteriorhodopsin or mutatedforms have been encapsulated in sol-gel matrices, which are opticallytransparent. Weetall et al. Biochem Biophys. Acta 1142:211-13 (1993); Wuet al. Chem. Mater. 5:115-20 (1993). Phycobiliproteins have also beenencapsulated. Chen et al. J. Sol-Gel Sci. & Tech. 7:99-108 (1996).Antibodies against small organic antigens have been encapsulated withinthe sol-gel. Bronshtein et al. Chem. Mater. 9:2632-39 (1997); Turnianskyet al. J. Sol-Gel Sci. & Tech. 7:135-43 (1996). Wang et al. Chem. Mater.65:2671-75 (1993). Alternatively, antigens have been encapsulated. Rouxet al. J. Sol-Gel Sci. & Tech. 8:663-66 (1997); Livage et al. J. Sol-GelSci. & Tech. 7:45-51 (1996). Biologics having novel magnetic properties,such as ferritin have been encapsulated. Lan et al. J. Sol-Gel Sci. &tech. 7:109-116 (1996). In certain embodiments, the reaction centerencapsulated need not be substantially purified from its natural source.Bresslar et al. J. Sol-Gel Sci. & Tech. 7:129-33 (1996).

Cells and organisms have been encapsulated in silica-based sol-gelmatrices. Peterson et al. P.S.E.B.M. 218:365-69 (1998). Bacteria havebeen immobilized in sol-gel matrices to metabolize herbicides forenvironmental clean-ups. Rietti-Shati et al. J. Sol-Gel Sci. & Tech.7:77-79 (1996). In using cells in a subject, it may be important toaccustom them to the implantation site before implantation to improvetheir viability. Differences in conditions such as glucoseconcentration, oxygen availability, nutrient concentrations, in vitroand in vivo may have an adverse affect on implantation of cells. Seegenerally U.S. Pat. Nos. 5,550,050; 5,620,883.

In addition to biological reaction centers, a variety of other materialshave been encapsulated in silica-based sol-gels. For example, organicfluorescent dyes and photochromic information recording materials havebeen encapsulated. Avnir et al. J. Phys. Chem. 88:5956-59 (1984); Avniret al. Journal of Non-Crystalline Solids, 74:395-406 (1985); Levy etal., Journal of Non-Crystalline Solids, 113:137-45 (1989). It is alsopossible to entrap a reaction center in a sol-gel for use in organiccatalysis. For example, lipases may be encapsulated in a sol-gel for useas a heterogeneous biocatalyst. Reetz et al. Angew. Chem. Int. Ed. Engl.34:301-03 (1995).

For those embodiments of the present invention that involveadministration of a sol-gel matrix to a subject, the reaction centerencapsulated therein need not be native to the subject. The sol-gelmatrix, as discussed above, may be immunoisolatory itself or modified tomake it so.

Any number of different types of reaction centers may be encapsulated ina single matrix. By encapsulating more than type of reaction center in asingle matrix, certain embodiments of the present invention may causethe conversion of one compound into a second and then into a third, andso on. Yamanka et al. J. Sol-Gel Sci. & Tech. 7:117-21 (1996); Chang etal. Artif. Organs 3:38-41 (1979). Such a result may be advantageous if,for example, the biological activity of the second compound isundesirable. Alternatively, it may be the case that it is the thirdcompound that has a more valuable biological activity than the second.Encapsulating more than one reaction center may increase the activity ofthe second reaction center in any pathway. For instance, the localconcentration of reactants for the second center may be increasedbecause of the reactivity of the first center. Fossel et al. Eur. J.Biochem. 30:165-71 (1987). Alternatively, for example, if the first typeof reaction center catalyzes an oxidation or reduction, the second typeof reaction center could mediate electron transfer and therebyfacilitate greater catalytic activity. For instance, theelectron-transfer redox pair Cc:CcP complex has been encapsulated. Linet al. J. Sol-Gel Sci. & Tech. 7:19-26 (1996).

In addition to reaction of the reaction center with a single othercompound, e.g., a prodrug, the present invention also contemplates thereaction of more than one material with the encapsulated reactioncenter. For example, it has been shown that NADP (nicotinamide adeninedinucleotide phosphate ester) may react with the reaction center andanother molecule. In one example, D-glucose-6-phosphate was converted byglucose-6-phosphate-dehydrogenase to a oxidized byproduct with theconcomitant reduction of NADP⁺ to NADPH. Yamanaka et al., J. Am. Chem.Soc. 117:9095-96 (1995). The types of reaction centers which may beencapsulated in a sol-gel, as contemplated by the present invention,thus includes those compounds or materials that react with more than onereactant. For example, many enzymes contemplated by the presentinvention for encapsulation require coenzymes in addition to anysubstrate.

One important characteristic of any encapsulated reaction center is thedegree of loading of the matrix. For example, the degree of loading mayaffect the reactivity of a reaction center in the sol-gel matrix. It hasbeen reported that the activity of trypsin appeared to decrease withincreased loading levels. Levels of loading contemplated by the presentinvention include 0.001, 0.01, 0.1, 1, 3, 5, 10, 15, 20 weight percentreaction center(s) and/or any additives to the matrix.

In certain embodiments, the present invention contemplates using as areaction center enzymes or other biological materials that are isolatedfrom, or otherwise substantially free of other cellular proteins. Theterm “substantially free of other cellular proteins” (also referred toherein as “contaminating proteins”) or “substantially pure or purifiedpreparations” are defined as encompassing preparations the reactioncenter of interest having less than 20% (by dry weight) contaminatingprotein, and preferably having less than 5% contaminating protein. Theterm “purified” as used herein preferably means at least 80% by dryweight, more preferably in the range of 95-99% by weight, and mostpreferably at least 99.8% by weight, of biological macromolecules of thesame type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above. “Isolated” and“purified” do not encompass either natural materials in their nativestate or natural materials that have been separated into components(e.g., in an acrylamide gel) but not obtained either as pure (e.g.lacking contaminating proteins, or chromatography reagents such asdenaturing agents and polymers, e.g. acrylamide or agarose) substancesor solutions.

In certain embodiments, the present invention contemplates using for thereaction center human homologs of any of the enzymes or other biologicalmaterials described herein, as well as orthologs and paralogs (homologs)in other species. The term “ortholog” refers to proteins which arehomologs via speciation, e.g., closely related and assumed to havecommon descent based on structural and functional considerations.Orthologous proteins function as recognizably the same activity indifferent species. The term “paralog” refers to genes or proteins whichare homologs via gene duplication, e.g., duplicated variants of a genewithin a genome. See also Fritch Syst Zool 19:99-113 (1970).

In certain embodiments, the present invention contemplates homologs ofany naturally occurring enzymes. Further, the present inventioncontemplates modification of the structure of any enzyme to enhancetherapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelflife). Such modified peptides may be produced, for instance, by aminoacid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In similar fashion, the amino acid repertoirecan be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine. (see, for example,Biochemistry (2nd ed., Stryer et al. eds. 1981). Whether a change in theamino acid sequence of a peptide results in a functional homolog to anynaturally occurring enzyme (e.g., functional in the sense that theresulting polypeptide mimics the wild-type form) can be readilydetermined by assaying for activity. Polypeptides in which more than onereplacement has taken place can readily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of any nucleic acid encoding for an enzyme used asa reaction center, as well as truncation mutants, and is especiallyuseful for identifying potential variant sequences (e.g. homologs) thatare functional in modulating the enzymatic activity of interest. Thepurpose of screening such combinatorial libraries is, for example, toidentify novel polypeptides that may convert prodrugs to biologicallyactive agents. In certain embodiments, such novel polypeptides mayconvert prodrugs that naturally occurring enzymes do not, which maytherefore allow a particular prodrug to be used in the present inventionthat otherwise would not have been possible. In other embodiments, theprodrug of interest is converted only by an encapsulated, novelpolypeptide. As a result, no biologically active agent is produced invivo except by the encapsulated reaction center of the matrix. Such aspecificity difference may have value because prodrugs that becomecyotoxic agents may have reduced toxicity if they are stable in vivo.Site-directed mutagenesis has been used in ADEPT to prepare mutants ofcarboxypeptidase A, so that only the mutants and no naturally occurringenzyme converts selected prodrugs into corresponding cytotoxic agents.Smith et al. J. Biol. Chem. 272:15804-16 (1997). Even a single aminoacid change may be sufficient to affect the specificity. Thus,combinatorially-derived homologs can be generated to have an increasedpotency or different specificity relative to a naturally occurring formof an enzyme or other biological macromolecule.

In one aspect of this method, the amino acid sequences for a populationof homologs for any enzyme, or other related proteins, are aligned,preferably to promote the highest homology possible. Such a populationof variants can include, for example, homologs from one or more species.Amino acids which appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences. In apreferred embodiment, the variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level, and is encoded by avariegated gene library. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins containing the set of sequences therein.

There are many ways by which such libraries of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then ligated into an appropriateexpression vector. The purpose of a degenerate set of genes is toprovide, in one mixture, all of the sequences encoding the desired setof potential sequences. The synthesis of degenerate oligonucleotides iswell known in the art. See, for example, Narang Tetrahedron 39:3 (1983);Itakura et al. Recombinant DNA. Proc 3rd Cleveland Sympos.Macromolecules 73-89 (ed. AG Walton, Amsterdam: Elsevier 1981); Itakuraet al. Annu. Rev. Biochem. 53:323 (1984); Itakura et al. Science198:1056 (1984); Ike et al. Nucleic Acid Res. 11:477 (1983). Suchtechniques have been employed in the directed evolution of otherproteins. See, for example, Scott et al. Science 249:386-90 (1990);Roberts et al. PNAS 89:2429-33 (1992); Devlin et al. Science 249:404-06(1990); Cwirla et al. PNAS 87:6378-82 (1990); as well as U.S. Pat. Nos.5,223,409, 5,198,346, and 5,096,815.

Likewise, a library of coding sequence fragments can be provided for anenzyme of interest as a reaction center in order to generate avariegated population of fragments for screening and subsequentselection of bioactive fragments. A variety of techniques are known inthe art for generating such libraries, including chemical synthesis. Inone embodiment, a library of coding sequence fragments can be generatedby (i) treating a double stranded PCR fragment of an coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule; (ii) denaturing the double stranded DNA; (iii) renaturingthe DNA to form double stranded DNA which can include sense/antisensepairs from different nicked products; (iv) removing single strandedportions from reformed duplexes by treatment with S1 nuclease; and (v)ligating the resulting fragment library into an expression vector. Bythis exemplary method, an expression library can be derived which codesfor N-terminal, C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of homologs for any enzyme of interest as a reaction center.The most widely used techniques for screening large gene librariestypically comprises cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors, and expressing the combinatorial genes under conditions inwhich detection of a desired activity facilitates relatively easyisolation of the vector encoding the gene whose product was detected.

5.4.1. Enzymes used for ADEPT

Enzymes that have been used for ADEPT may generally be used as theencapsulated reaction center in the present invention. Such enzymes wereoriginally chosen because they convert a prodrug into a biologicallyactive agent, and they are thereby of use in the present invention aswell. Some examples of such enzymes, and the conversion that theycatalyze, follow. Other examples may be found in U.S. Pat. Nos.5,714,148; 5,660,829, 5,587,161; and 5,405,990. In the presentinvention, enzymes used in ADEPT need not necessarily be used with thesame prodrug as used in the ADEPT application.

In one aspect of the invention, a variety of peptidases, which cleaveamide bonds, may be used as the reaction center. Jungheim et al., Chem.Rev. 94:1553-66 (1994). In one embodiment, carboxypeptidase G2 can beused as the reaction center with prodrugs to cleave an amide bond. Onebiologically active agent is nitrogen mustard, which is an alkylatingagent. Carboxypeptidase G2 cleaves an amide bond of a prodrug to givethe free nitrogen mustard and glutamic acid. Bagshawe, Br. J. Cancer56:531 (1987); Bagshawe et al. Br. J. Cancer 58:700 (1988). In anotherembodiment, carboxypeptidase A from bovine pancrease can be used as thereaction center with prodrugs to cleave an amide bond. In oneembodiment, the biologically active agent is methotrexate, which is usedfor cancer chemotherapy, and the prodrugs are α-peptides ofmethotrexate, e.g., Glu-α-L-Ala-methotrexate andL-Glu-L-Phe-methotrexate. Vitols et al., Pteridines 3:125 (1992);Kuefner et al., Biochem. 28:2288 (1989); Haenseler et al., Biochem.31:891 (1992).

In another embodiment, penicillin V amidase from Fusarium oxysporum canbe used as the reaction center with prodrugs to deacylate an N-acylamine. In several embodiments, the biologically active agent isdoxorubicin or mephalan, which are anticancer agents, and the prodrugsare N-acyl derivatives of doxorubicin or mephalan. Kerr et al. CancerImmun. Immunother. 31:202 (1990). In another embodiment, penicillin Gamidase can be used as the reaction center with prodrugs to cleavephenylacetamides. In several embodiments, the biologically active agentis doxorubicin or mephalan, and the prodrugs are phenylacetamidederivatives of doxorubicin or mephalan. Kerr et al. supra. In anotherembodiment, the biologically active agent is palytoxin, which is apotent cytotoxin, and the prodrug isN-[(4′-hydroxyphenyl)acetyl]palytoxin. Because palytoxin asserts itseffect extracellularly, it may be able to overcome the multidrugresistance phenotype.

If another embodiment, urokinase may be the reaction center. Puromycinand doxorubicin have been produced using this enzyme. WO 91/09134. Inanother embodiment, a variety of β-lactamases, which cleave certainamide bonds, may be used as the reaction center. In one embodiment, abiologically active agent is produced by covalently attaching the agentto the C-3′ position of cephalosporin or a derivative of cephalosporin,whereupon hydrolization of the prodrug by a β-lactamase, e.g., P99enzyme derived from Enterobacter cloacae 265A or enzyme derived from B.cereus, produces the free agent. Biologically active agents that havebeen covalently attached to cephalosporin or a derivative ofcephalosporin in this manner include: methotrexate; 5-fluorouracil,which is often used for the treatment of colon cancer; LY233425, apotent analogue of the anticancer agent vinblastine;desacetylvinblastine hydrazine, a potent vinca alkaloid; nitrogenmustard alkylating agents; thioguanine; doxorubicin; mitomycin C; andDACCP, a carboplatinum-based drug that is a potent antitumor agent.Jungheim et al. Chem. Rev. 94:1553-66 (1994); Meyer et al. Antibody.Immunoconjugates, Radiopharm. 3:66 (1990); Jungheim et al., Antibody,Immunoconjugates. Radiopharm. 4:228 (1991); Shepard et al., Biomed.Chem. Lett. 1:21 (1991); EP0382411A2; Alexander et al. Tetrahedron Lett.32:3269 (1991); EPo392745A2; Svensson et al. Bioconj. Chem. 3:176(1992); Vrudhula et al. Bioconj. Chem. 4:334 (1993); Hudyma et al.Biomed. Chem. Lett. 3:323 (1993); EP0484870A2; Junghein et al.Heterocycles 35:33 (1993); Hanessian et al. Can J. Chem. 71:896 (1993).

In another aspect of the invention, alkaline phosphotase can be used asthe reaction center with prodrugs to remove phosphate from organicphosphates. Biologically active agents that are produced in this mannerinclude etoposide, mitomycin-derived agents, nitrogen mustard derivedagents, and doxorubicin. Senter et al. Proc. Nat. Acad. Sci. U.S.A.85:4842 (1988); Senter et al. Cancer Res. 49:5789 (1989); Senter, FASEBJ. 4:188 (1990); Sahin et al. Cancer Res. 50:6944 (1990).

In another aspect of the invention, glycosidases, which cleave aglycosidic linkage may be used as the reaction center. In oneembodiment, β-glucuronidase is used as the reaction center to producebiologically active agents, including nitrogen mustard derived agents,daunomycin, adriamycin, epirubicin. Roffler et al. Biomed Pharmacol.42:2062 (1991); Wang et al. Cancer Res. 52:4484 (1992); Deonarain et al.Br. J. Cancer 70:786-94 (1994). In another embodiment, β-glucosidaseconverts amygdalin into glucose, benzaldehyde, and hydrogen cyanide, atoxic species. In another embodiment, α-galactosidase is used as thereaction center to produce daunorubicin. Andrianomenjanahary et al.Biomed. Chem. Lett. 2:1093 (1992).

In another aspect of the invention, cytosine deaminase, which convertscytosine into uracil, may be used as the reaction center. In oneembodiment, cytosine deaminase isolated from Bakers' yeast is used toproduce the antitumor agent 5-fluroruracil from 5-fluorocytosine. Senteret al. Bioconj. Chem. 2:447 (1991). In another aspect of the invention,nitroreductase, which requires the presence of a cofactor such as NADH,may be used as the reaction center. The enzyme has been used to produce5-aziridin-4-hydroxyamino-2-nitrobenzamide from5-aziridin-2,4-dinitrobenzamide. Knox et al. Cancer Metathesis Rev.12:195 (1993).

In another aspect of the invention, oxidases, which produce reducedoxygen species, e.g., peroxide, superoxide, and hydroxyl radicals, maybe used as the reaction center. In one embodiment, glucose oxidase andlactoperoxidase convert glucose and iodide into hydrogen peroxide andtoxic iodine species. Ito et al. Bone Marrow Transplant. 6:395-98(1990); Stanislawski (1989). In another embodiment, xathine oxidaseproduces reduced oxygen species from either xanthine or hypoxanthine.Dinota et al. Bone Marrow Transplant. 6:31-36 (1990).

5.4.2. Other Enzymes

In addition to the enzymes used for ADEPT, for which a prodrug may havealready been identified, other enzymes may be used as the reactioncenter. In using any enzyme in the present invention, it may benecessary to consider which compounds will be converted by the enzyme.Which enzyme is most suitable for encapsulation as the reaction centerdepends, in part, on the expected use of any matrix.

In deciding which enzyme may be appropriate for any application, thegeneral classification of enzymes may be used in identifying andconsidering different types of reactions that a reaction center couldpossibly catalyze. These classes include: (i) oxidoreductases (acting onthe CH—OH group of donors; acting on the aldehyde or oxo group ofdonors; acting on the CH—CH group of donors; acting on the CH—NH(2)group of donors; acting on the CH—NH group of donors; acting on NADH orNADPH; acting on other nitrogenous compounds as donors; acting on asulfur group of donors; acting on a heme group of donors; acting ondiphenols and related substances as donors; acting on a peroxide asacceptor (peroxidases); acting on hydrogen as donor; acting on singledonors with incorporation of molecular oxygen; acting on paired donorswith incorporation of molecular oxygen; acting on superoxide radicals asacceptor; oxidizing metal ions; acting on —CH(2) groups; acting onreduced ferredoxin as donor; acting on reduced flavodoxin as donor;other oxidoreductases); (ii) transferases (transferring one-carbongroups; transferring aldehyde or ketone residues; acyltransferases;glycosyltransferases; transferring alkyl or aryl groups, other thanmethyl groups; transferring nitrogenous groups; transferringphosphorous-containing groups; transferring sulfur-containing groups;transferring selenium-containing groups); (iii) hydrolases (acting onester bonds; glycosidases; acting on ether bonds; acting on peptidebonds (peptide hydrolases); acting on carbon-nitrogen bonds, other thanpeptide bonds; acting on acid anhydrides; acting on carbon-carbon bonds;acting on halide bonds; acting on phosphorus-nitrogen bonds; acting onsulfur-nitrogen bonds; acting on carbon-phosphorus bonds; acting onsulfur-sulfur bonds); lyases (carbon-carbon lyases; carbon-oxygenlyases; carbon-nitrogen lyases; carbon-sulfur lyases; carbon-halidelyases; phosphorus-oxygen lyases; other lyases); (iv) isomerases(racemases and epimerases; cis-trans-isomerases; intramolecularoxidoreductases; intramolecular transferases (mutases); intramolecularlyases; other isomerases); (v) ligases (forming carbon-oxygen bonds;forming carbon-sulfur bonds; forming carbon-nitrogen bonds; formingcarbon-carbon bonds; forming phosphoric ester bonds).

Illustrative examples of enzymes that may serve as reaction centers inthe present invention include, without limitation: alcohol dehydrogenase(EC 1.1.1.1), homoserine dehydrogenase (EC 1.1.1.3), (RR)-butanedioldehydrogenase (EC 1.1.1.4), glycerol dehydrogenase (EC 1.1.1.6),glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8), D-xylulosereductase (EC 1.1.1.9), L-xylulose reductase (EC 1.1.1.10), L-iditoldehydrogenase (EC 1.1.1.14), mannitol-1-phosphate dehydrogenase (EC1.1.1.17), myo-inositol 2-dehydrogenase (EC 1.1.1.18), aldehydereductase (EC 1.1.1.21), quinate dehydrogenase (EC 1.1.1.24), shikimatedehydrogenase (EC 1.1.1.25), glyoxylate reductase (EC 1.1.1.26),L-lactate dehydrogenase (EC 1.1.1.27), D-lactate dehydrogenase (EC1.1.1.28), glycerate dehydrogenase (EC 1.1.1.29), 3-hydroxybutyratedehydrogenase (EC 1.1.1.30), 3-hydroxyisobutyrate dehydrogenase (EC1.1.1.31), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), malatedehydrogenase (EC 1.1.1.37), malate dehydrogenase and aspartateaminotransferase (EC 1.1.1.37 & 2.6.1.1), malate dehydrogenase andcitrate (si)-synthase (EC 1.1.1.37 and 4.1.3.7), malate dehydrogenase(decarboxylating) (EC 1.1.1.39), malate dehydrogenase(oxaloacetate-decarboxylating) (NADP+) (EC 1.1.1.40), isocitratedehydrogenase (NADP+) (EC 1.1.1.42), phosphogluconate dehydrogenase(decarboxylating) (EC 1.1.1.44), glucose dehydrogenase (EC 1.1.1.47),galactose dehydrogenase (EC 1.1.1.48), glucose-6-phosphate dehydrogenase(EC 1.1.1.49), glucose-6-phosphate dehydrogenase and6-phosphogluconolactonase (EC 1.1.1.49 & 3.1.1.31), 3-hydroxysteroiddehydrogenase (EC 1.1.1.50), 3(or 17)-hydroxysteroid dehydrogenase (EC1.1.1.51), lactaldehyde reductase (NADPH) (EC 1.1.1.55), ribitoldehydrogenase (EC 1.1.1.56), 3-hydroxypropionate dehydrogenase (EC1.1.1.59), 2-hydroxy-3-oxopropionate reductase (EC 1.1.1.60),4-hydroxybutyrate dehydrogenase (EC 1.1.1.61), estradiol17-dehydrogenase (EC 1.1.1.62), mannitol dehydrogenase (EC 1.1.1.67),gluconate 5-dehydrogenase (EC 1.1.1.69), glycerol dehydrogenase (NADP+)(EC 1.1.1.72), glyoxylate reductase (NADP+) (EC 1.1.1.79), aryl-alcoholdehydrogenase (EC 1.1.1.90), phosphoglycerate dehydrogenase (EC1.1.1.95), diiodophenylpyruvate reductase (EC 1.1.1.96),3-hydroxybenzyl-alcohol dehydrogenase (EC 1.1.1.97),3-oxoacyl-[acyl-carrier-protein] reductase (EC 1.11.100), carnitinedehydrogenase (EC 1.1.1.108), indolelactate dehydrogenase (EC1.1.1.110), glucose dehydrogenase (NADP+) (EC 1.1.1.119), fructose5-dehydrogenase (NADP+) (EC 1.1.1.124), 2-deoxy-D-gluconatedehydrogenase (EC 1.1.1.125), L-threonate dehydrogenase (EC 1.1.1.129),sorbitol-6-phosphate dehydrogenase (EC 1.1.1.140),15-hydroxyprostaglandin dehydrogenase (NAD+) (EC 1.1.1.141),21-hydroxysteroid dehydrogenase (NAD+) (EC 1.1.1.150), sepiapterinreductase (EC 1.1.1.153), coniferyl-alcohol dehydrogenase (EC1.1.1.194), (R)-2-hydroxyglutarate dehydrogenase (EC 1.1.1.a),sorbitol-6-phosphate dehydrogenase (NADP+) (EC 1.1.1.b), gluconate2-dehydrogenase (EC 1.1.99.3), lactate-malate transhydrogenase (EC1.1.99.7), glucoside 3-dehydrogenase (EC 1.1.99.13), formatedehydrogenase (EC 1.2.1.2), acetaldehyde dehydrogenase (acetylating) (EC1.2.1.10), aspartate-semialdehyde dehydrogenase (EC 1.2.1.11),glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12),glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (EC1.2.1.12 & 2.7.2.3), glyoxylate dehydrogenase (acylating) (EC 1.2.1.17),formate dehydrogenase (NADP+) (EC 1.2.1.43), succinate dehydrogenase (EC1.3.99.1), butyryl-CoA dehydrogenase (EC 1.3.99.2), dihydroorotatedehydrogenase (EC 1.3.99.11), alanine dehydrogenase (EC 1.4.1.1),glutamate dehydrogenase (EC 1.4.1.2), glutamate dehydrogenase (NAD(P)+)(EC 1.4.1.3), glutamate dehydrogenase (NADP+) (EC 1.4.1.4), leucinedehydrogenase (EC 1.4.1.9), glycine dehydrogenase (EC 1.4.1.10),L-erytlro-3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11),2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12),pyrroline-2-carboxylate reductase (EC 1.5.1.1), pyrroline-5-carboxylatereductase (EC EC 1.5.1.2), dihydrofolate reductase (EC EC 1.5.1.3),methylenetetrahydrofolate dehydrogenase (NADP+) (EC 1.5.1.5), D-octopinedehydrogenase (EC 1.5.1.11), methylenetetrahydrofolate dehydrogenase(NAD+) (EC 1.5.1.15), alanopine dehydrogenase (EC 1.5.1.17),1-piperidine-2-carboxylate reductase (EC 1.5.1.21),NAD(P)+transhydrogenase (EC 1.6.1.1), glutathione reductase (NAD(P)H)(EC 1.6.4.2), thioredoxin reductase (NADPH) (EC 1.6.4.5), NADHdehydrogenase (EC 1.6.99.3), 5,10-methylenetetrahydrofolate reductase(FADH2) (EC 1.7.99.5), dihydrolipoamide dehydrogenase (EC 1.8.1.4),glutathione-CoA-glutathione transhydrogenase (EC 1.8.4.3), cytochrome-coxidase (EC EC 1.9.3.1), hydrogen dehydrogenase (EC 1.12.1.2),thetin-homocysteine S-methyltransferase (EC 2.1.1.3), homocysteineS-methyltransferase (EC 2.1.1.10), thymidylate synthase (EC 2.1.1.45),glycine hydroxymethyltransferase (EC 2.1.2.1), glycineformiminotransferase (EC 2.1.2.4), glutamate formiminotransferase (EC2.1.2.5), D-alanine 2-hydroxymethyltransferase (EC 2.1.2.7),aminomethyltransferase (EC 2.1.2.10), methylmalonyl-CoAcarboxyltransferase (EC 2.1.3.1), ornithine carbamoyltransferase (EC2.1.3.3), oxamate carbamoyltransferase (EC 2.1.3.5), glycineamidinotransferase (EC 2.1.4.1), transketolase (EC 2.2.1.1),transaldolase (EC 2.2.1.2), imidazole N-acetyltransferase and phosphateacetyltransferase (EC 2.3.1.2 & 2.3.1.8), arylamine N-acetyltransferase(EC 2.3.1.5), choline O-acetyltransferase (EC 2.3.1.6), camitineO-acetyltransferase (EC 2.3.1.7), phosphate acetyltransferase (EC2.3.1.8), phosphate acetyltransferase and formate C-acetyltransferase(EC 2.3.1.8 & 2.3.1.54), phosphate acetyltransferase and acetate kinase(EC 2.3.1.8 & 2.7.2.1), acetyl-CoA C-acetyltransferase (EC 2.3.1.9),carnitine O-palmitoyltransferase (EC 2.3.1.21), glutamateN-acetyltransferase (EC 2.3.1.35), [acyl-carrier-protein]S-acetyltransferase (EC 2.3.1.38), [acyl-carrier-protein]S-malonyltransferase (EC 2.3.1.39), formate C-acetyltransferase (EC2.3.1.54), sucrose phosphorylase (EC 2.4.1.7), maltose phosphorylase (EC2.4.1.8), levansucrase (EC 2.4.1.10), sucrose synthase (EC 2.4.1.13),sucrose-phosphate synthase (EC 2.4.1.14), -trehalose-phosphate synthase(UDP-forming) (EC 2.4.1.15), cellobiose phosphorylase (EC 2.4.1.20),laminaribiose phosphorylase (EC 2.4.1.31), -trehalose phosphorylase (EC2.4.1.64), galactinol-raffinose galactosyltransferase (EC 2.4.1.67),sinapate 1-glucosyltransferase (EC 2.4.1.120), purine-nucleosidephosphorylase (EC 2.4.2.1), purine-nucleoside phosphorylase andpyrimidine-nucleoside phosphorylase (EC 2.4.2.1 & 2.4.2.2),pyrimidine-nucleoside phosphorylase (EC 2.4.2.2), uridine phosphorylase(EC 2.4.2.3), nucleoside deoxyribosyltransferase (EC 2.4.2.6), adeninephosphoribosyltransferase (EC 2.4.2.7), hypoxanthinephosphoribosyltransferase (EC 2.4.2.8), orotatephosphoribosyltransferase (EC 2.4.2.10), guanosine phosphorylase (EC2.4.2.15), thiamine pyridinylase (EC 2.5.1.2), thiamin-phosphatepyrophosphorylase (EC 2.5.1.3), aspartate transaminase (EC 2.6.1.1),malate dehydrogenase and aspartate transaminase (EC 1.1.1.37 & 2.6.1.1),alanine transaminase (EC 2.6.1.2), histidinol-phosphate transaminase (EC2.6.1.9), ornithine-oxo-acid transaminase (EC 2.6.1.13),glutamine-pyruvate aminotransaminase (EC 2.6.1.15),succinyldiaminopimelate transaminase (EC 2.6.1.17), -alanine-pyruvatetransaminase (EC 2.6.1.18), 4-aminobutyrate transaminase (EC 2.6.1.19),D-alanine transaminase (EC 2.6.1.21), pyridoxamine-pyruvate transaminase(EC 2.6.1.30), dTDP-4-amino-4,6-dideoxy-D-glucose transaminase (EC2.6.1.33), glycine-oxaloacetate transaminase (EC 2.6.1.35),2-aminoadipate transaminase (EC 2.6.1.39), serine-pyruvate transaminase(EC 2.6.1.51), phosphoserine transaminase (EC 2.6.1.52), hexokinase (EC2.7.1.1), galactokinase (EC 2.7.1.6), 6-phosphofructokinase (EC2.7.1.11), NAD+kinase (EC 2.7.1.23), dephospho-CoA kinase (EC 2.7.1.24),glycerol kinase (EC 2.7.1.30), protein kinase (EC 2.7.1.37), pyruvatekinase (EC 2.7.1.40), 1-phosphatidylinositol kinase (EC 2.7.1.67),pyrophosphate-serine phosphotransferase (EC 2.7.1.80),pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90),acetate kinase (EC 2.7.2.1), carbamate kinase (EC 2.7.2.2),phosphoglycerate kinase (EC 2.7.2.3), glyceraldehyde-3-phosphatedehydrogenase and phosphoglycerate kinase (EC 1.2.1.12 & 2.7.2.3),aspartate kinase (EC 2.7.2.4), guanidinoacetate kinase (EC 2.7.3.1),creatine kinase (EC 2.7.3.2), creatine kinase and myosin ATPase (EC2.7.3.2 & 3.6.1.32), arginine kinase (EC 2.7.3.3), taurocyamine kinase(EC 2.7.3.4), lombricine kinase (EC 2.7.3.5), phosphomevalonate kinase(EC 2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase(EC 2.7.4.4), nucleoside-diphosphate kinase (EC 2.7.4.6), guanylatekinase (EC 2.7.4.8), nucleoside-triphosphate-adenylate kinase (EC2.7.4.10), (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13), cytidylatekinase (EC 2.7.4.14), ribose-plosphate pyrophosphokinase (EC 2.7.6.1),nicotinamide-nucleotide adenylyltransferase (EC 2.7.7.1), sulfateadenylyltransferase (EC 2.7.7.4), sulfate adenylyltransferase andinorganic pyrophosphatase (EC 2.7.7.4 & 3.6.1.1), DNA-directed DNApolymerase (EC 2.7.7.7), UTP-glucose-1-phosphate uridylyltransferase (EC2.7.7.9), UDP glucose-hexose-1-phosphate uridylyltransferase (EC2.7.7.12), UDPglucose-hexose I-phosphate uridylyltransferase and UDPglucose (EC 2.7.7.12 & 5.1.3.2), mannose-1-phosphate guanylyltransferase(EC 2.7.7.13), ethanolamine-phosphate cytidylyltransferase (EC2.7.7.14), choline-phosphate cytidylyltransferase (EC 2.7.7.15),UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23),glucose-1-phosphate thymidylyltransferase (EC 2.7.7.24),glucose-1-phosphate adenylyltransferase (EC 2.7.7.27),glucose-1-phosphate cytidylyltransferase (EC 2.7.7.33),glucose-1-phosphate guanylyltransferase (EC 2.7.7.34),[glutamate-ammonia-ligase] adenylyltransferase (EC 2.7.7.42),glucuronate-1-phosphate uridylyltransferase (EC 2.7.7.44), pyruvate,orthophosphate dikinase (EC 2.7.9.1), aryl sulfotransferase (EC2.8.2.1), 3-oxoacid CoA-transferase (EC 2.8.3.5), acetateCoA-transferase (EC 2.8.3.8), triacylglycerol lipase (EC 3.1.1.3),acetylcholinesterase (EC 3.1.1.7), retinyl-palmitate esterase (EC3.1.1.21), glucose-6-phosphate dehydrogenase and6-phosphogluconolactonase (EC 1.1.1.49 & 3.1.1.31), alkaline phosphatase(EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), phosphoserine phosphatase(EC 3.1.3.3), 5′-nucleosidase (EC 3.1.3.5), fructose-biphosphatase (EC3.1.3.11), phosphodiesterase I (EC 3.1.4.1), 3′,5′-cyclic-nucleotidephosphodiesterase (EC 3.1.4.17), phosphohydrolase (unclassified) (EC3.1.4.a), ribonuclease T2 (EC 3.1.27.1), pancreatic ribonuclease (EC3.1.27.5), ribonuclease (unclassified) (EC 3.1.27.a), cyclomaltodextringlucanotransferase and -amylase (EC 2.4.1.19 & 3.2.1.1), -amylase (EC3.2.1.2), glucan 1,4-glucosidase (EC 3.2.1.3), oligo-1,6-glucosidase (EC3.2.1.10), -glucosidase (EC 3.2.1.20), -glucosidase (EC 3.2.1.21),-galactosidase (EC 3.2.1.23), -mannosidase (EC 3.2.1.24),-fructofuranosidase (EC 3.2.1.26), -dextrin endo-1,6-glucosidase (EC3.2.1.41), AMP nucleosidase (EC 3.2.2.4), NAD+ nucleosidase (EC3.2.2.5), NAD(P)+ nucleosidase (EC 3.2.2.6), adenosine nucleosidase (EC3.2.2.7), adenosylhomocysteinase (EC 3.3.1.1), leucyl aminopeptidase (EC3.4.11.1), dipeptidyl-peptidase (EC 3.4.14.1), carboxypeptidase A (EC3.4.17.1), gly-X carboxypeptidase (EC 3.4.17.4), -glu-X carboxypeptidase(EC 3.4.19.9), chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), papain(EC 3.4.22.2), pepsin A (EC 3.4.23.1), chymosin (EC 3.4.23.4),thermolysin (EC 3.4.24.27), asparaginase (EC 3.5.1.1), glutaminase (EC3.5.1.2), urease (EC 3.5.1.5), penicillin amidase (EC 3.5.1.11),aminoacylase (EC 3.5.1.14), pantothenase (EC 3.5.1.22),N-methyl-2-oxoglutaramate hydrolase (EC 3.5.1.36), dihydroorotase (EC3.5.2.3), carboxymethylhydantoinase (EC 3.5.2.4), -lactamase (EC3.5.2.6), arginase (EC 3.5.3.1), allantoicase (EC 3.5.3.4), argininedeiminase (EC 3.5.3.6), adenosine deaminase (EC 3.5.4.4), cytidinedeaminase (EC 3.5.4.5), AMP deaminase (EC 3.5.4.6),methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), inorganicpyrophosphatase (EC 3.6.1.1), sulfate adenylyltransferase and inorganicpyrophosphatase (EC 2.7.7.4 & 3.6.1.1), trimetaphosphatase (EC 3.6.1.2),nucleotide pyrophosphatase (EC 3.6.1.9), myosin ATPase (EC 3.6.1.32),creatine kinase and myosin ATPase (EC 2.7.3.2 & 3.6.1.32),Ca2+-transporting ATPase (EC 3.6.1.38), chymotrypsin (EC 3.4.21.1),thermolysin (EC 3.4.24.4), phosphoenolpyruvate carboxykinase (GTP) (EC4.1.1.32), phosphoenolpyruvate carboxykinase (diphosphate) (EC4.1.1.38), ribulose-biphosphate carboxylase (EC 4.1.1.39),ketotetraose-phosphate aldolase (EC 4.1.2.2), deoxyribose-phosphatealdolase (EC 4.1.2.4), fructose-biphosphate aldolase (EC 4.1.2.13),fructose-biphosphate aldolase and triose-phosphate isomerase (EC4.1.2.13 & 5.3.1.1), 2-dehydro-3-deoxyphosphogluconate aldolase (EC4.1.2.14), L-fuculose-phosphate aldolase (EC 4.1.2.17),2-dehydro-3-deoxy-L-pentonate aldolase (EC 4.1.2.18),rhamulose-1-phosphate aldolase (EC 4.1.2.19),2-dehydro-3-deoxy-6-phosphogalactonate aldolase (EC 4.1.2.21),D-arabino-3-hexulose phosphate formaldehyde lyase (EC 4.1.2.a),isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2),N-acetylneuraminate lyase (EC 4.1.3.3), citrate (pro-3S)-lyase (EC4.1.3.6), citrate (si)-synthase (EC 4.1.3.7), citrate (si)-synthase andmalate dehydrogenase (EC 4.1.3.7 & 1.1.1.37), ATP citrate(pro-3S)-lyase(EC 4.1.3.8), 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16),citramalate lyase (EC 4.1.3.22), malyl-CoA lyase (EC 4.1.3.24),2,3-dimethylmalate lyase (EC 4.1.3.32), tryptophanase (EC 4.1.99.1),fumarate hydratase (EC 4.2.1.2), aconitate hydratase (EC 4.2.1.3),3-dehydroquinate dehydratase (EC 4.2.1.10), phosphopyruvate hydratase(EC 4.2.1.11), enoyl-CoA hydratase (EC 4.2.1.17), tryptophan synthase(EC 4.2.1.20), maleate hydratase (EC 4.2.10.31), (S)-2-methylmalatedehydratase (EC 4.2.1.34), (R)-2-methylmalate dehydratase (EC 4.2.1.35),D-glutamate cyclase (EC 4.2.1.48), urocanate hydratase (EC 4.2.1.49),crotonoyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58),dimethylmaleate hydratase (EC 4.2.1.85), 3-hydroxybutyryl-CoA dehyratase(EC 4.2.1.a), aspartate ammonia-lyase (EC 4.3.1.1), methylaspartateammonia-lyase (EC 4.3.1.2), histidine ammonia-lyase (EC 4.3.1.3),phenylalanine ammonia-lyase (EC 4.3.1.5), -alanyl-CoA ammonia lyase (EC4.3.1.6), arginosuccinate lyase (EC 4.3.2.1), adenylosuccinate lyase (EC4.3.2.2), ureidoglycolate lyase (EC 4.3.2.3), lactoylglutathione lyase(EC 4.4.1.5), adenylate cyclase (EC 4.6.1.1), alanine racemase (EC5.1.1.1), glutamate racemase (EC 5.1.1.3), lysine racemase (EC 5.1.1.5),diaminopimelate epimerase (EC 5.1.1.7), 4-hydroxyproline epimerase (EC5.1.1.8), amino-acid racemase (EC 5.1.1.10), ribulose-phosphate3-epimerase (EC 5.1.3.1), UDP glucose 4-epimerase (EC 5.1.3.2), UDPglucose 4-epimerase and UDPglucose-hexose 1-phosphate (EC 5.1.3.2 &2.7.7.12), L-ribulose-phosphate 4-epimerase (EC 5.1.3.4), UDP arabinose4-epimerase (EC 5.1.3.5), UDP glucuronate 4-epimerase (EC 5.1.3.6),N-acylglucosamine 2-epimerase (EC 5.1.3.8),N-acylglucosamine-6-phosphate 2-epimerase (EC 5.1.3.9), CDPabequoseepimerase (EC 5.1.3.10), glucose-6-phosphate 1-epimerase (EC 5.1.3.15),GDP-D-mannose 3,5-epimerase (EC 5.1.3.18), methylmalonyl-CoA epimerase(EC 5.1.99.1), retinal isomerase (EC 5.2.1.3), linoleate isomerase (EC5.2.1.5), triose-phosphate isomerase (EC 5.3.1.1), triose-phosphateisomerase and fructose-bisphosphate aldolase (EC 5.3.1.1 & 4.1.2.13),erythrose isomerase (EC 5.3.1.2), arabinose isomerase (EC 5.3.1.3),L-arabinose isomerase (EC 5.3.1.4), xylose isomerase (EC 5.3.1.5),ribose-5-phosphate isomerase (EC 5.3.1.6), mannose isomerase (EC5.3.1.7), mannose-6-phosphate isomerase (EC 5.3.1.8),glucose-6-phosphate isomerase (EC 5.3.1.9), glucosamine-6-phosphateisomerase (EC 5.3.1.10), glucuronate isomerase (EC 5.3.1.12),arabinose-5-phosphate isomerase (EC 5.3.1.13), L-rharinose isomerase (EC53.1.14), D-lyxose ketol-isomerase (EC 5.3.1.15), ribose isomerase (EC5.3.1.20), L-mannose ketol-isomerase (EC 5.3.1.a),phospho-3-hexuloisomerase (EC 5.3.1.b), phenylpyruvate tautomerase (EC5.3.2.1), oxaloacetate tautomerase (EC 5.3.2.2),isopentenyl-diphosphate-isomerase (EC 5.3.3.2),methylitaconate-isomerase (EC 5.3.3.6), phosphoglycerate mutase (EC5.4.2.1), phosphoglucomutase (EC 5.4.2.2), phosphoacetylglucosaminemutase (EC 5.4.2.3), -phosphoglucomutase (EC 5.4.2.6),phosphopentomutase (EC 5.4.2.7), phosphomannomutase (EC 5.4.2.8), lysine2,3-aminomutase (EC 5.4.3.2), D-ornithine 4,5-aminomutase (EC 5.4.3.5),methylaspartate mutase (EC 5.4.99.1), methylmalonyl-CoA mutase (EC5.4.99.2), 2-methyleneglutarate mutase (EC 5.4.99.4), muconatecycloisomerase (EC 5.5.1.1), tetrahydroxypteridine cycloisomerase (EC5.5.1.3), chalcone isomerase (EC 5.5.1.6), valine-tRNA ligase (EC6.1.1.9), acetate-CoA ligase (EC 6.2.1.1), butyrate-CoA ligase (EC6.2.1.2), succinate-CoA ligase (GDP-forming) (EC 6.2.1.4), succinate-CoAligase (ADP forming) (EC 6.2.1.5), glutamate-ammonia ligase (EC6.3.1.2), formate-tetrahydrofolate ligase (EC 6.3.4.3), adenylosuccinatesynthase (EC 6.3.4.4), arginosuccinate synthase (EC 6.3.4.5), pyruvatecarboxylase (EC 6.4.1.1), propanoyl-CoA carboxylase (EC 6.4.1.3),hemoglobin, tyrosine hydroxylase, prohonnone convertase, bcl-2, dopadecarboxylase, and dopamine beta-hydroxylase.

5.4.3. Assay of Encapsulated Reaction Centers

Any number of methods are available to determine whether a reactioncenter retains its ability to convert prodrug to biologically activeagent upon encapsulation. Those of skill in the art will be able tomodify, if necessary, any standard procedures developed for assaying thereaction center in free solution for assaying the encapsulated reactioncenter. For example, if the matrix is transparent, as is true forsilica-based sol-gel matrices, then standard visible and UV-Vistechniques for solid materials may be employed. Yamanka et al. J.Sol-Gel sci. & tech. 7:117-21 (1996). If the reaction center is redoxactive center, e.g., a transition metal, then other spectroscopies, suchas EPR, may be employed. Lin et al. J. Sol-Gel Sci. & Tech. 7:19-26(1996). As discussed below for the prodrugs, assaying for reactioncenter activity often involves measuring the reactants and products, andsolution techniques may be applicable. Alternatively, coenzymes,cofactors, or other reactants involved in any reaction center may bemonitored as an assay for activity of any encapsulated reaction center.

From such assays, it should be possible to determine the reactionkinetics for the encapsulated reaction center. In general, for enzymes,the reaction kinetics may correspond to the Michaelis-Menten treatment.Zubay et al. Biochemistry 137-141 (1983). An apparent Michaelis constant(Km) may be determined for the encapsulated reaction center. Dosoretz etal., J. Sol-Gel Sci. & Tech. 7:7-11 (1996). The Km for thenonencapsulated reaction center and the Km of the encapsulated reactioncenter may be compared. In certain embodiments, the ratio of Km(nonencapsulated) to Km (encapsulated) may be greater than one. Dosoretzet al., supra. In other embodiments, the ratio may be less than one.Venton et al. Biochim Biophys Acta 1250:117-25 (1995). The presentinvention contemplates ratios of 100, 10, 5, 1, 0.5, 0.1, 0.005, 0.01,and 0.001. Of course, for determining any of these ratios, theconditions of the reaction should be kept as similar as possible.

The encapsulation of reaction centers allows for the design of novelassays for reaction center activity. For example, a second reactioncenter may be encapsulated so as to help assay the activity of a firstencapsulated reaction center. Yamanka et al. report encapsulating bothoxalate oxidase and peroxidase. The peroxidase converts two dyeprecursors into a detectable dye using hydrogen peroxide, which isformed by oxalate oxidase from oxalate, water, and dioxygen. Yamanka etal. J. Sol-Gel Sci. & Tech. 7:117-21 (1996). Hence, the peroxidase inthis sol-gel matrix assists in assaying the reaction kinetics of theoxalate oxidase. Yamanka et al. report that this enzyme system is usefulas a diagnostic for the decreased secretion of oxalate in cases ofhyperglycinemia, hypoclycinuria, and hyperoxaluria. Ngo et al. Anal.Biochem. 105:389 (1980).

5.5. Prodrugs

5.5.1. Prodrugs Contemplated by the Invention

A variety of materials or compounds may be employed as prodrugs in thepresent invention. A number of such prodrugs have been discussedelsewhere, including when considering possible reaction centers and usesof the present invention. Any compound that is biologically active maybe used in the present invention as a prodrug, as long as a suitableprodrug may be prepared that may be converted by a reaction center intoa biologically active compound. The matrices of the present inventionmay be administered by way of oral ingestion or implantation. Ifimplantation is desired, they can be implanted subcutaneously,constitute a part of a prosthesis, or be inserted in a cavity of thehuman body. Subcutaneous implantation using a syringe consists ofinjecting the amtrices directly into subcutaneous tissue. Thus, thematrices of the present invention can be suspended in a physiologicalbuffer and introduced via a syringe to the desired site. In certaincases, a biologically active agent may itself be used as a prodrug inthe present invention if a reaction center modulates its biologicalactivity upon reaction.

A number of considerations may be weighed by those of skill in the artin determining which prodrug is appropriate for any use of the presentinvention. For example, it may be necessay to match a prodrug with areaction center that has a high activity for conversion of the prodrug.Alternatively, in choosing a prodrug, it may be important to considerwhere in a subject the resulting matrix may be administered, e.g., theuse of the prodrug L-dopa to produce the biologically active agentdopamine in the striatum. Another possible consideration may be thephysical dimension of any prodrug, for to operate as a prodrug, it mayneed to diffuse into the matrix for conversion by the reaction center toa biologically active agent. (Alternatively, the reaction center may belocated on the surface of the matrix, whereupon no diffusion isnecessary for conversion of the prodrug.) However, even antibodies havebeen observed to diffuse into matrices of the present invention, so anyprodrug of at least that dimension may be used in the present invention.As discussed in preparing the matrices of the present invention, it maybe necessary to ensure that the physical size of the reaction center isgreater than that of its counterpart prodrug so as to prevent leaching.This criteria need not always apply, however, because for example, thereaction center may be covalently attached to the matrix, which mayprevent any substantial leaching, or alternatively, any leaching thatmay occur may be acceptable for any use that the matrix is put.

Possible biologically active agents, which may be used as prodrugs inthe present invention after appropriate modification, include withoutlimitation, medicaments; vitamins; mineral supplements; substances usedfor the treatment, prevention, diagnosis, cure or mitigation of diseaseor illness; or substances which affect the structure or function of thebody.

Specific types of biologically active agents include, withoutlimitation: anti-angiogenesis factors, antiinfectives such asantibiotics and antiviral agents; analgesics and analgesic combinations;anorexics; antihelmintics; antiarhritics; antiasthmatic agents;anticonvulsants; antidepressants; antidiuretic agents; antidiarrheals;antihistamines; antiinflammatory agents; antimigraine preparations;antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics;antipsychotics; antipyretics, antispasmodics; anticholinergics;sympathomimetics; xanthine derivatives; cardiovascular preparationsincluding calcium channel blockers and beta-blockers such as pindololand antiarrhythmics; antihypertensives; catecholamines; diuretics;vasodilators including general coronary, peripheral and cerebral;central nervous system stimulants; cough and cold preparations,including decongestants; growth factors, hormones such as estradiol andother steroids, including corticosteroids; hypnotics;immunosuppressives; muscle relaxants; parasympatholytics;psychostimulants; sedatives; and tranquilizers; and naturally derived orgenetically engineered proteins, polysaccharides, glycoproteins,lipoproteins, interferons, cytokines, chemotherapeutic agents and otheranti-neoplastics, antibiotics, anti-virals, anti-fungals,anti-inflammatories, anticoagulants, lymphokines, or antigenicmaterials.

To illustrate further, other types of biologically active agents thatmay be used as prodrugs upon appropriate modification if necessary,including peptide, proteins or other biopolymers, e.g., interferons,interleukins, tumor necrosis factor, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), glialcell line-derived neurotrophic factor (GDNF), cholinergicdifferentiation factor/Leukemia inhibitory factor (CDF/LIF), epidermalgrowth factor (EGF), insulin-like growth factor (IGF), basic fibroblastgrowth factor (bFGF), platelet-derived growth factor (PDGF),erythropoietin, growth hormone, Substance-P, neurotensin, insulin,erythropoietin, albumin, transferrin, and other protein biologicalresponse modifiers.

Other examples of biologically active agents that may be used asprodrugs in accordance with the present invention either directly orafter appropriate modification include acebutolol, acetaminophen,acetohydoxamic acid, acetophenazine, acyclovir, adrenocorticoids,allopurinol, alprazolam, aluminum hydroxide, amantadine, ambenonium,amiloride, aminobenzoate potassium, amobarbital, amoxicillin,amphetamine, ampicillin, androgens, anesthetics, anticoagulants,anticonvulsants-dione type, antithyroid medicine, appetite suppressants,aspirin, atenolol, atropine, azatadine, bacampicillin, baclofen,beclomethasone, belladonna, bendroflumethiazide, benzoyl peroxide,benzthiazide, benztropine, betamethasone, betha nechol, biperiden,bisacodyl, bromocriptine, bromodiphenhydramine, brompheniramine,buclizine, bumetanide, busulfan, butabarbital, butaperazine, caffeine,calcium carbonate, captopril, carbamazepine, carbenicillin, carbidopa &levodopa, carbinoxamine inhibitors, carbonic anhydsase, carisoprodol,carphenazine, cascara, cefaclor, cefadroxil, cephalexin, cephradine,chlophedianol, chloral hydrate, chlorambucil, chloramphenicol,chlordiazepoxide, chloroquine, chlorothiazide, chlorotrianisene,chlorpheniramine, 6× chlorpromazine, chlorpropamide, chlorprothixene,chlorthalidone, chlorzoxazone, cholestyramine, cimetidine, cinoxacin,clemastine, clidinium, clindamycin, clofibrate, clbmiphere, clonidine,clorazepate, cloxacillin, colochicine, coloestipol, conjugated estrogen,contraceptives, cortisone, cromolyn, cyclacillin, cyclandelate,cyclizine, cyclobenzaprine, cyclophosphamide, cyclothiazide, cycrimine,cyproheptadine, danazol, dantlhron, dantrolene, dapsone,dextroamphetamine, dexamethasone, dexchlorpheniramine, dextromethorphan,diazepan, dicloxacillin, dicyclomine, diethylstilbestrol, diflunisal,digitalis, diltiazen, dimenhydrinate, dimethindene, diphenhydramine,diphenidol, diphenoxylate & atrophive, diphenylopyraline, dipyradamole,disopyramide, disulfiram, divalporex, docusate calcium, docusatepotassium, docusate sodium, doxyloamine, dronabinol ephedrine,epinephrine, ergoloidmesylates, ergonovine, ergotamine, erythromycins,esterified estrogens, estradiol, estrogen, estrone, estropipute,etharynic acid, ethchlorvynol, ethinyl estradiol, ethopropazine,ethosaximide, ethotoin, fenoprofen, ferrous fumarate, ferrous gluconate,ferrous sulfate, flavoxate, flecainide, fluphenazine, fluprednisolone,flurazepam, folic acid, furosemide, gemfibrozil, glipizide, glyburide,glycopyrrolate, gold compounds, griseofuwin, guaifenesin, guanabenz,guanadrel, guanethidine, halazepam, haloperidol, hetacillin,hexobarbital, hydralazine, hydrochlorothiazide, hydrocortisone(cortisol), hydroflunethiazide, hydroxychloroquine, hydroxyzine,hyoscyamine, ibuprofen, indapamide, indomethacin, insulin, iofoquinol,iron-polysaccharide, isoetharine, isoniazid, isopropamide isoproterenol,isotretinoin, isoxsuprine, kaolin & pectin, ketoconazole, lactulose,levodopa, lincomycin liothyronine, liotrix, lithium, loperamide,lorazepam, magnesium hydroxide, magnesium sulfate, magnesiumtrisilicate, maprotiline, meclizine, meclofenamate, medroxyproyesterone,melenamic acid, melphalan, mephenyloin, mephobarbital, meprobamate,mercaptopurine, mesoridazine, metaproterenol, metaxalone,methamphetamine, methaqualone, metharbital, methenamine, methicillin,methocarbamol, methotrexate, methsuximide, methyclothinzide,methylcellulos, methyldopa, methylergonovine, methylphenidate,methylprednisolone, methysergide, metoclopramide, metolazone,metoprolol, metronidazole, minoxidil, mitotane, monamine oxidaseinhibitors, nadolol, nafcillin, nalidixic acid, naproxen, narcoticanalgesics, neomycin, neostigmine, niacin, nicotine, nifedipine,nitrates, nitrofurantoin, nomifensine, norethindrone, norethindroneacetate, norgestrel, nylidrin, nystatin, orphenadrine, oxacillin,oxazepam, oxprenolol, oxymetazoline, oxyphenbutazone, pancrelipase,pantothenic acid, papaverine, para-aminosalicylic acid, paramethasone,paregoric, pemoline, penicillamine, penicillin, penicillin-v,pentobarbital, perphenazine, phenacetin, phenazopyridine, pheniramine,phenobarbital, phenolphthalein, phenprocoumon, phensuximide,phenylbutazone, phenylephrine, phenylpropanolamine, phenyl toloxamine,phenyloin, pilocarpine, pindolol, piper acetazine, piroxicam, poloxamer,polycarbophil calcium, polythiazide, potassium supplements, pruzepam,prazosin, prednisolone, prednisone, primidone, probenecid, probucol,procainamide, procarbazine, prochlorperazine, procyclidine, promazine,promethazine, propantheline, propranolol, pseudoephedrine, psoralens,psyllium, pyridostigmine, pyrodoxine, pyrilamine, pyrvinium, quinestrol,quinethazone, quinidine, quinine, ranitidine, rauwolfia alkaloids,riboflavin, rifampin, ritodrine, salicylates, scopolamine, secobarbital,senna, sannosides a & b, simethicone, sodium bicarbonate, sodiumphosphate, sodium fluoride, spironolactone, sucrulfate, sulfacytine,sulfamethoxazole, sulfasalazine, sulfinpyrazone, sulfisoxazole,sulindac, talbutal, tamazepam, terbutaline, terfenadine, terphinhydrate,teracyclines, thiabendazole, thiamine, thioridazine, thiothixene,thyroblobulin, thyroid, thyroxine, ticarcillin, timolol, tocainide,tolazamide, tolbutamide, tolmetin trozodone, tretinoin, triamcinolone,trianterene, triazolam, trichlormethiazide, tricyclic antidepressants,tridhexethyl, trifluoperazine, triflupromazine, trihexyphenidyl,trimeprazine, trimethobenzamine, trimethoprim, tripclennamine,triprolidine, valproic acid, verapamil, vitamin A, vitamin B-12, vitaminC, vitamin D, vitamin E, vitamin K, xanthine, parathyroid hormone,enkephalins, and endorphins.

To illustrate further, antimetabolites may be used as prodrugs uponappropriate modification if necessary, including without limitationmethotrexate, 5-fluorouracil, cytosine arabinoside (ara-C),5-azacytidine, 6-mercaptopurine, 6-thioguanine, and fludarabinephosphate. Antitumor antibiotics may include but are not limited todoxorubicin, daunorubicin, dactinomycin, bleomycin, mitomycin C,plicamycin, idarubicin, and mitoxantrone. Vinca alkaloids andepipodophyllotoxins may include, but are not limited to vincristine,vinblastine, vindesine, etoposide, and teniposide. Nitrosoureas,including carmustine, lomustine, semustine and streptozocin, may also beprodrugs, upon appropriate modification if necessary. Hormonaltherapeutics may also be prodrugs, upon appropriate modification ifnecessary, such as corticosteriods (cortisone acetate, hydrocortisone,prednisone, prednisolone, methyl prednisolone and dexamethasone),estrogens, (diethylstibesterol, estradiol, esterified estrogens,conjugated estrogen, chlorotiasnene), progestins (medroxyprogesteroneacetate, hydroxy progesterone caproate, megestrol acetate),antiestrogens (tamoxifen), aromastase inhibitors (aminoglutethimide),androgens (testosterone propionate, methyltestosterone, fluoxymesterone,testolactone), antiandrogens (flutamide), LHRH analogues (leuprolideacetate), and endocrines for prostate cancer (ketoconazole). Antitumordrugs that are radiation enhancers may also be used as prodrugs, uponappropriate modification if necessary. Examples of such biologicallyactive agents include, for example, the chemotherapeutic agents5′-fluorouracil, mitomycin, cisplatin and its derivatives, taxol,bleomycins, daunomycins, and methamycins. Antibiotics may be used asprodrugs as well, upon appropriate modification if necessary, and theyare well known to those of skill in the art, and include, for example,penicillins, cephalosporins, tetracyclines, ampicillin, aureothicin,bacitracin, chloramphenicol, cycloserine, erythromycin, gentamicin,gramacidins, kanamycins, neomycins, streptomycins, tobramycin, andvancomycin.

Other prodrugs, upon appropriate modification if necessary, which may beused in the present invention include those presently classified asinvestigational drugs, and can include, but are not limited toalkylating agents such as Nimustine AZQ, BZQ, cyclodisone, DADAG,CB10-227, CY233, DABIS maleate, EDMN, Fotemustine, Hepsulfam,Hexamethylmelamine, Mafosamide, MDMS, PCNU, Spiromustine, TA-077, TCNUand Temozolomide; antimetabolites, such as acivicin, Azacytidine,5-aza-deoxycytidine, A-TDA, Benzylidene glucose, Carbetimer, CB3717,Deazaguanine mesylate, DODOX, Doxifluridine, DUP-785, 10-EDAM,Fazarabine, Fludarabine, MZPES, MMPR, PALA, PLAC, TCAR, TMQ, TNC—P andPiritrexim; antitumor antibodies, such as AMPAS, BWA770U, BWA773U,BWA502U, Amonafide, m-AMSA, C₁₋₉₂₁, Datelliptium, Mitonafide,Piroxantrone, Aclarubicin, Cytorhodin, Epirubicin, esorubicin,Idarubicin, Iodo-doxorubicin, Marcellomycin, Menaril, Morpholinoanthracyclines, Pirarubicin, and SM-5887; microtubule spindleinhibitors, such as Amphethinile, Navelbine, and Taxol; thealkyl-lysophospholipids, such as BM41-440, ET-18-OCH₃, andHexacyclophosphocholine; metallic compounds, such as Gallium Nitrate,CL286558, CL287110, Cycloplatam, DWA2114R, NK121, Iproplatin,Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium compounds; andnovel compounds such as, for example, Aphidoicolin glycinate, Ambazone,BSO, Caracemide, DSG, Didemnin, B, DMFO, Elsamicin, Espertatrucin,Flavone acetic acid, HMBA, HHT, ICRF-187, Iododeoxyuridine, Ipomeanol,Liblomycin, Lonidamine, LY186641, MAP, MTQ, Merabarone SK&F104864,Suramin, Tallysomycin, Teniposide, THU and WR2721; and Toremifene,Trilosane, and zindoxifene.

5.5.2. Assays and Identification of Prodrugs.

As a general matter, it will be clear to one of skill in the art whichprodrugs may be used with which reaction centers so as to effect the anyof the uses of the subject invention, e.g., producing a biologicallyactive agent. Prodrugs that display desired characteristics, e.g.,certain kinetic profiles of conversion of a prodrug by a reaction centerto the corresponding biologically active agent, may serve as leadcompounds for the discovery of more desirable prodrugs.

In general, there are a number of methods by which useful prodrugs ofany reaction center encapsulated in a sol gel may be determined. Forexample, prodrugs may be individually prepared and tested for productionof the corresponding biologically active agent upon interaction with thereaction center, whether encapsulated or not.

In another embodiment of the present invention, the use of prodrugs inthis invention readily lends itself to the creation of combinatoriallibraries of compounds for screening prospective prodrugs with anyparticular reaction center or group of reaction centers to identifyprodrugs of such reaction centers. For the purposes of the presentinvention, the application of combinatorial chemistry may be especiallyvaluable because it may render identification of a suitable prodrug of abiologically active agent for use with a particular reaction center morefacile. A combinatorial library for the purposes of the presentinvention is a mixture of chemically related compounds which may bescreened together for a desired property, e.g., conversion by aparticular reaction center or reaction centers to produce a biologicallyactive agent. Such libraries may be in solution or covalently linked toa solid support. The preparation of many related compounds asprospective prodrugs in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate reactivity of any prospective prodrug maybe done by conventional methods.

For purposes of this invention, diversity in a library may be created ata variety of different levels. In general, for instance, substrate arylgroups used in a combinatorial approach can be diverse in terms of thecore aryl moiety, e.g., a variegation in terms of the ring structure,and/or can be varied with respect to the other substituents. Withrespect to the subject invention, for example, it is generally knownthat carboxypeptidases hydrolyze amide bonds. Any biologically activeagent having an amine or carboxylic acid moiety may, in theory, bederivatized in a combinatorial approach to form an amide with acarboxylic acid or amine moiety, respectively. For example, a peptidylfragment having a varied number of amino acid residues with a diverseidentity could be coupled to a biologically active agent of interest togive a library of prospective prodrugs, whereupon conversion by reactioncenters such as carboxypeptidases of the prospective prodrugs in thelibrary could be screened. In this fashion, prospective prodrugs of abiologically active agent could be prepared and screened for use with aparticular reaction center, or alternatively, a group of reactioncenters that catalyze a similar type of chemical conversion. As alreadynoted above, the reaction centers themselves may be prepared andscreened by combination methods as well. As will be clear to one ofskill in the art, in preparing any such library, some considerations totake into account include the chemical conversion catalyzed by thereaction center or centers of interest; in what fashion a biologicallyactive agent may be readily derivatized to provide prodrugs so that thereaction center or centers of interest could produce the biologicallyactive agent from a prodrug; and the specificity of the reaction centeror centers of interest to a variation in structure with respect to thereaction that it normally catalyzes, e.g., the naturally occurringsubstrate for an enzyme.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See generallyBlondelle et al. Trends Anal. Chem. 14:83 (1995); U.S. Pat. Nos.5,359,115, 5,362,899, 5,288,514, and 5,721,099; Chen et al. JACS116:2661 (1994; Kerr et al. JACS 115:252 (1993); WO92/10092, WO93/09668,WO 94/08051, WO93/20242 and WO91/07087. Accordingly, a variety oflibraries on the order of about 16 to 1,000,000 or more diversomers canbe synthesized and screened for a particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can besynthesized using the subject reactions adapted to the techniquesdescribed in WO 94/08051, e.g., being linked to a polymer bead by ahydrolyzable or photolyzable group, e.g., located at one of thepositions of substrate. According to the technique disclosed therein,the library is synthesized on a set of beads, each bead including a setof tags identifying the particular diversomer on that bead. In oneembodiment, the beads can be dispersed on the surface of a permeablemembrane, and the diversomers released from the beads by lysis of thebead linker. The diversomer from each bead will diffuse across themembrane to an assay zone, where it will interact with an assay for areaction center or centers. Detailed descriptions of a number ofcombinatorial methodologies are provided below.

(a) Direct Characterization. A growing trend in the field ofcombinatorial chemistry is to exploit the sensitivity of techniques suchas mass spectrometry (MS), e.g., which can be used to characterizesub-femtomolar amounts of a compound, and to directly determine thechemical constitution of a compound selected from a combinatoriallibrary. For instance, where the library is provided on an insolublesupport matrix, discrete populations of compounds can be first releasedfrom the support and characterized by MS. In other embodiments, as partof the MS sample preparation technique, such MS techniques as MALDI canbe used to release a compound from the matrix, particularly where alabile bond is used originally to tether the compound to the matrix. Forinstance, a bead selected from a library can be irradiated in a MALDIstep in order to release the diversomer from the matrix, and ionize thediversomer for MS analysis.

(b) Multipin Synthesis. The libraries of the subject method can take themultipin library format. Briefly, Geysen and co-workers, Geysen et al.PNAS 81:3998-4002 (1984), introduced a method for generating compoundlibraries by a parallel synthesis on polyacrylic acid-gratedpolyethylene pins arrayed in the microtitre plate format. The Geysentechnique can be used to synthesize and screen thousands of compoundsper week using the multipin method, and the tethered compounds may bereused in many assays. Appropriate linker moieties can also beenappended to the pins so that the compounds may be cleaved from thesupports after synthesis for assessment of purity and furtherevaluation. Compare Bray et al. Tetrahedron Lett. 31:5811-14 (1990);Valerio et al. Anal Biochem 197:168-77 (1991); Bray et al. TetrahedronLett. 32:6163-66 (1991).

(c) Divide-Couple-Recombine. In yet another embodiment, a variegatedlibrary of compounds can be provided on a set of beads utilizing thestrategy of divide-couple-recombine. See, for example, Houghten PNAS82:5131-35 (1985); and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971.Briefly, as the name implies, at each synthesis step where degeneracy isintroduced into the library, the beads are divided into separate groupsequal to the number of different substituents to be added at aparticular position in the library, the different substituents coupledin separate reactions, and the beads recombined into one pool for thenext iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags. Houghten et al. PNAS 82:5131-35(1986). Substituents are coupled to the compound-bearing resins byplacing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

(d) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis. A scheme of combinatorial synthesis inwhich the identity of a compound is given by its locations on asynthesis substrate is termed a spatially-addressable synthesis. In oneembodiment, the combinatorial process is carried out by controlling theaddition of a chemical reagent to specific locations on a solid support.Dower et al. Annu Rep Med Chem 26:271-280 (1991); Fodor, Science 251:767(1991); U.S. Pat. No. 5,143,854; Jacobs et al. Trends Biotechnol12:19-26 (1994). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al. J MedChem 37:1233-51 (1994). A synthesis substrate is prepared for couplingthrough the covalent attachment of photolabile nitroveratryloxycarbonyl(NVOC) protected amino linkers or other photolabile linkers. Light isused to selectively activate a specified region of the synthesis supportfor coupling. Removal of the photolabile protecting groups by light(deprotection) results in activation of selected areas. Afteractivation, the first of a set of amino acid analogs, each bearing aphotolabile protecting group on the amino terminus, is exposed to theentire surface. Coupling only occurs in regions that were addressed bylight in the preceding step. The reaction is stopped, the plates washed,and the substrate is again illuminated through a second mask, activatinga different region for reaction with a second protected building block.The pattern of masks and the sequence of reactants define the productsand their locations. Since this process utilizes photolithographytechniques, the number of compounds that can be synthesized is limitedonly by the number of synthesis sites that can be addressed withappropriate resolution. The position of each compound is preciselyknown; hence, its interactions with other molecules can be directlyassessed. With respect to the above example, for example, a library ofpeptidyl fragments could thereby be prepared, whereupon the biologicallyactive agent could be coupled in the final step to produce a diverselibrary of prospective prodrugs when paired with reaction centers thathydrolyze peptide bonds.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

(e) Encoded Combinatorial Libraries. In yet another embodiment, thesubject method utilizes a compound library provided with an encodedtagging system. A recent improvement in the identification of activecompounds from combinatorial libraries employs chemical indexing systemsusing tags that uniquely encode the reaction steps a given bead hasundergone and, by inference, the structure it carries. Conceptually,this approach mimics phage display libraries, where activity derivesfrom expressed peptides, but the structures of the active peptides arededuced from the corresponding genomic DNA sequence. The first encodingof synthetic combinatorial libraries employed DNA as the code. A varietyof other forms of encoding have been reported, including encoding withsequenceable bio-oligomers (e.g., oligonucleotides and peptides), andbinary encoding with additional non-sequenceable tags.

(1) Tagging with sequenceable bio-oligomers. The principle of usingoligonucleotides to encode combinatorial synthetic libraries wasdescribed in 1992 Brenner et al. PNAS 89:5381-83 (1992), and an exampleof such a library appeared the following year. Needles et al. PNAS90:10700-04 (1993). A combinatorial library of nominally 77 (=823,543)peptides composed of all combinations of Arg, Gin, Phe, Lys, Val, D-Valand Thr (three-letter amino acid code), each of which was encoded by aspecific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), wasprepared by a series of alternating rounds of peptide andoligonucleotide synthesis on solid support. In this work, the aminelinking functionality on the bead was specifically differentiated towardpeptide or oligonucleotide synthesis by simultaneously preincubating thebeads with reagents that generate protected OH groups foroligonucleotide synthesis and protected NH₂ groups for peptide synthesis(here, in a ratio of 1:20). When complete, the tags each consisted of69-mers, 14 units of which carried the code. The bead-bound library wasincubated with a fluorescently labeled antibody, and beads containingbound antibody that fluoresced strongly were harvested byfluorescence-activated cell sorting (FACS). The DNA tags were amplifiedby PCR and sequenced, and the predicted peptides were synthesized.Following such techniques, compound libraries can be derived for use inthe subject method, where the oligonucleotide sequence of the tagidentifies the sequential combinatorial reactions that a particular beadunderwent, and therefore provides the identity of the compound on thebead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach, Kerret al. J Am Chem Soc 115:2529-31 (1993), orthogonality in synthesis isachieved by employing acid-labile protection for the coding strand andbase-labile protection for the compound strand.

In an alternative approach, Nikolaiev et al. Pept Res 6:161-70 (1993),branched linkers are employed so that the coding unit and the testcompound can both be attached to the same functional group on the resin.In one embodiment, a cleavable linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and the compound. Ptek et al. Tetrahedron Lett 32:3891-94 (1991).In another embodiment, the cleavable linker can be placed so that thetest compound can be selectively separated from the bead, leaving thecode behind. This last construct is particularly valuable because itpermits screening of the test compound without potential interference ofthe coding groups. Examples in the art of independent cleavage andsequencing of peptide library members and their corresponding tags hasconfirmed that the tags can accurately predict the peptide structure.

(2) Non-sequenceable Tagging: Binary Encoding. An alternative form ofencoding the test compound library employs a set of non-sequencableelectrophoric tagging molecules that are used as a binary code. Ohlmeyeret al. PNAS 90:10922-26 (1993). Exemplary tags are haloaromatic alkylethers that are detectable as their trimethylsilyl ethers at less thanfemtomolar levels by electron capture gas chromatography (ECGC).Variations in the length of the alkyl chain, as well as the nature andposition of the aromatic halide substituents, permit the synthesis of atleast 40 such tags, which in principle can encode 2⁴⁰ (e.g., upwards of10¹²) different molecules. In the original report, Ohlmeyer et al.,supra, the tags were bound to about 1% of the available amine groups ofa peptide library via a photocleavable o-nitrobenzyl linker. Thisapproach is convenient when preparing combinatorial libraries ofpeptide-like or other amine-containing molecules. A more versatilesystem has, however, been developed that permits encoding of essentiallyany combinatorial library. Here, the compound would be attached to thesolid support via the photocleavable linker and the tag is attachedthrough a catechol ether linker via carbene insertion into the beadmatrix. Nestler et al. J Org Chem 59:4723-24 (1994). This orthogonalattachment strategy permits the selective detachment of library membersfor assay in solution and subsequent decoding by ECGC after oxidativedetachment of the tag sets.

Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase, Ohlmeyer et al. PNAS 92:6027-31 (1995), andprovide guidance for generating the subject compound library Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

When prospective prodrugs are screened as libraries of compounds, highthroughput assays are desirable in order to maximize the number ofcompounds surveyed in a given period of time. The activity of thereaction center, e.g., enzymatic activity, with any prospective prodrugmay be determined by monitoring either the disappearance of prodrug orthe appearance of the corresponding biologically active agent.Alternatively, the reaction center activity may be determined bymonitoring the reduction or production or any reactants consumed orby-products produced. Spectroscopic methods well-known to those of skillmay be used for such monitoring, or alternatively, any of the reactantsor products, e.g., the prodrug or the corresponding biologically activeagent, may be isolated and quantified.

In other embodiments, differential assays can be used to identifyprodrugs that react more readily with a encapsulated reaction centerthan with any other naturally occurring enzyme, so that any such prodrugare converted chiefly by any administered encapsulated reaction centerinstead of by any naturally occurring enzymes or catalysts. Such afeature may be desirable depending on how the matrix is used.

5.6. Administration

5.6.1. Matrix Administration

Immobilized enzymes may be administered in a variety of ways. Seegenerally Ming et al. Methods for Therapeutic Applications 46:676-699.The site of administration of the matrix may affect its therapeuticeffect depending on the reaction center encapsulated therein. Forexample, the site of implantation of encapsulated PCI2 cells fortreatment of Parkinson's disease appears to affect the device output.Emerich et al. Cell Transplant. 5:589-96 (1996).

A number of different implantation sites in a subject are contemplatedfor the matrices of this invention. In particular, the most preferredsite is determined by the identity of the encapsulated reaction center.Any site that results in a therapeutic effect may be used. For example,for reaction centers that produce biologically active agents that arecytotoxic, the implants may be implanted near any neoplasm. ADEPTtechnology relies on such proximity to deliver any cytotoxic agentessentially directly to the tumor. In another instance, for matricesused to treat Parkinson's disease by affecting dopamine levels in thebrain, implantation in the brain may be preferred. Other sites in thebrain for such matrices include the basal ganglia, the substantia nigra,and the striatum.

The matrices of the present invention may be administered by way of oralingestion or implantation. If implantation is desired, they can beimplanted subcutaneously, constitute a part of a prosthesis, or beinserted in a cavity of the human body. Subcutaneous implantation usinga syringe consists of injecting the matrices directly into subcutaneoustissue. Thus, the matrices of the present invention can be suspended ina physiological buffer and introduced via a syringe to the desired site.Other sites include the central nervous system, including the brain,spinal cord, and aqueous and vitreous humors of the eye. Other sites inthe brain include the cerebral cortex, subthalamic nuclei and nucleusBasalis of Maynert. Other sites include the cerebrospinal fluid, thesubarachnoid space, and the lateral ventricles. Other sites includes thekidney subcapsular site, and intraperitoneal and subcutaneous sites.

In other embodiments of the present invention, the matrices of thepresent invention may be associated with a medical article to be used asan implant. For example, matrices of the present invention could beattached as thin films to such devices. Alternatively, matrices of thepresent invention could be attached as a capsule or incorporated intoany medical device. Exemplary structural medical articles include suchimplants as orthopedic fixation devices, ventricular shunts, laminatesfor degradable fabric, drug-carriers, burn dressings, coatings to beplaced on other implant devices, and the like.

For administration of matrices of the present invention, an importantfeature may be whether the matrix is intended to stay in place afteradministration or move in the subject. For example, matricesadministered to a subject may be transported and localized in thelymphatic system as part of the subject immune response to the foreignobjects.

Once a matrix of the present invention is administered, it may remain inat least partial contact with a biological fluid, such as blood,internal organ secretions, mucus membranes, cerebrospinal fluid, and thelike.

The length of the period during which encapsulated reaction centerremains active enough so as to produce a therapeutic effect may dependon a variety of features. Enzymes encapsulated in silica-based sol-gelmatrices have remained active for periods of several months. Theadministration of any matrix of the present invention may result in thelong-terin, stable production of a biologically active agent.

5.6.2. Formulations and Use of Matrices and Prodrugs

In addition to the general introduction, pharmaceutical compositions foruse in accordance with the present invention may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. Thus, as appropriate, matrices and any prodrug,including any physiologically acceptable salts and solvates, may beformulated for administration by, for example, injection, inhalation orinsulation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration. Appropriate formulations maydepend, in part, on the administration method used and whether a prodrugor a matrix is being administered.

The matrices or prodrugs of the invention may be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. Intramuscular, intravenous, intraperitoneal, andsubcutaneous injection is possible. For injection, the matrices orprodrugs of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the prodrugs may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the matrices or prodrugs may take the form of,for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). The tabletsmay be coated by methods well known in the art. Liquid preparations fororal administration may take the form of, for example, solutions, syrupsor suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of any prodrug. For buccal administration theprodrugs may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the prodrugs foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The prodrugs and/or matrices may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the prodrug may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The prodrugs may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the prodrugs andmatrices of the present invention may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the prodrugs may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.Other suitable delivery systems include microspheres which offer thepossibility of local noninvasive delivery of drugs over an extendedperiod of time. This technology utilizes microspheres of precapillarysize which can be injected via a coronary catheter into any selectedpart of the e.g. heart or other organs without causing inflammation orischemia. Other methods of controlled release of the prodrugs andmatrices of the present invention are known to those of skill in theart.

Systemic administration may also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the prodrugs of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

The prodrugs and/or matrices may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

The prodrugs may be employed in the present invention in various forms,such as molecular complexes or pharmaceutically acceptable salts.Representative examples of such salts are succinate, hydrochloride,hydrobromide, sulfate, phosphate, nitrate, borate, acetate, maleate,tartrate, salicylate, metal salts (e.g., alkali or alkaline earth),ammonium or amine salts (e.g., quaternary ammonium) and the like.Furthermore, derivatives of the prodrugs such as esters, amides, andethers which have desirable retention and release characteristics butwhich are readily hydrolyzed in vivo by physiological pH or enzymes canalso be employed.

5.7. Treatment

The selected dosage level for the matrices and prodrugs, if applicable,of the present invention will depend upon a variety of factorsincluding: the load of the reaction center within the matrix; theactivity of the reaction center, the activity of the particular prodrugemployed, or the ester, salt or amide thereof, the route ofadministration; the time of administration, the rate of excretion of theparticular prodrug (and possibly matrix) being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular matrix and prodrug employed, the age,sex, weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

Toxicity and therapeutic efficacy of the matrices of the presentinvention may be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅/ED₅₀. In certain embodiments, those inwhich an exogenous prodrug is activated by the reaction centerencapsulated in a biocompatible matrix of the subject invention, theefficacy of treatment using the subject invention may be gauged bycomparing any of the foregoing parameters resulting from treatment usinga prodrug alone (i.e., without the subject matrix), the biologicallyactive substance that results from the prodrug alone, and treatmentusing the prodrug and a subject matrix as disclosed herein. In certainembodiments, treatments using the subject invention have ratios of abouttwo (or less), five, ten, one hundred, one thousand or even greaterorders of magnitude more favorable than treatment with the prodrug aloneor the biologically active substance that results from the prodrugalone.

Because the matrix itself does not result in a therapeutic effectwithout the involvement of some other compound, e.g., a prodrug ornaturally occurring metabolite, but also the availability of othercompounds that interact with the matrix may affect any treatment regime.In general, matrices and the biologically active agents that theyproduce which exhibit large therapeutic indices are preferred. Bytargeting the matrix to a particular region of a subject so as tolocalize the production of the biologically active agent, thetherapeutic efficacy may be dramatically increased, and unwanted sideeffects may be minimized. For example, by implanting the dopamineproducing matrix in the striatum, it may not be necessary to administerL-dopa with carbidopa or benserazide, which is used to combat nausearesulting from conversion of L-dopa to dopamine outside of the brain.

Because the matrix, upon administration, may be in place and active forsignificant time periods, ant treatment regime may involve multipleadministrations of a prodrug so as to produce biologically active agent.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any matrix and/orprodrug used in the present invention, the therapeutically effectivedose can be estimated initially from cell culture assays. For example, adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma of abiologically active agent may be measured, for example, by highperformance liquid chromatography.

EXEMPLIFICATIONS

The present invention now being generally described, it may be morereadily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention in any way.

A. Reaction Center Encapsulation Studies

1. Matrix Preparation

The general synthetic technique used for preparation of the silica solwas addition of 21 mL of tetramethyl orthosilicate (Aldrich, 99+%) and5.08 mL of a 4 mM HCl solution to a 25×150 mm test tube equipped with astirbar. The mixture is stirred until homogeneous (approximately 15minutes). The test tube containing the sol is then transferred to an icebath and allowed to cool for 10 minutes. A 2 mL aliquot of sol is thentransferred to another chilled test tube in an ice bath and stirred. Tothis sol, 1 mL of chilled buffer solution (appropriate to the enzyme tobe entrapped) is added, and stirred for ca. 10s, followed by addition of1 mL of chilled, buffered solution containing the desired enzyme. Thesol is swirled briefly, and then pipetted into a 4.5 mL polystyrenecuvette (cell culture dishes were also used for surface area studymatrices). The cuvette opening is sealed with Parafilm following gelformation (cell culture dish covers were used for surface area studymatrices). The gel is then allowed to age in the sealed container for aperiod of time ranging from 18 h to 50 d or more at temperatures rangingfrom 4° C. to room temperature. Selected samples were dried at ambienttemperature over a period of days to weeks by puncturing the Parafilmcovering the container opening. Other samples were assayed withoutdrying.

2. Enzyme Encapsulation and Assays

a) b-Glucosidase

The entrapment of b-Glucosidase (from almonds, crude, lyophilizedpowder, Sigma) was performed as outlined above, using 50 mM, pH 5.0acetate buffer. The activity assay was performed using 2.667 mL of a 10mM solution of para-nitrophenyl-b-D-glucopyranoside (Sigma, 99+%) inbuffer and 37.33 mL acetate buffer in a 125 mL Erlenmeyer flask (40 mLtotal solution volume). 2 mL aliquots of solution were removed for assayand their UV-Vis spectra recorded.

b) Penicillinase

Entrapment of Penicillinase (Type I from Bacillus cereus, lyophilizedpowder containing approx. 10% protein, Sigma) was performed as outlinedabove, using 50 mM pH 6.5 phosphate buffer. Penicillinase activity wasdetermined using 100 mL of a 3 mM solution of Penicillin G(Benzylpenicillin, sodium salt, Sigma) in buffer. 2 mL aliquots of thereaction solution were removed for assay and their UV-Vis spectrarecorded.

c) Tyrosinase

Entrapment of Tyrosinase (from mushroom, Sigma) was performed asoutlined above, with 50 mM pH 6.5 phosphate buffer solution. Tyrosinaseactivity assays were performed using 0.3 mM L-tyrosine (Aldrich; 99+%)solution in buffer.

d) Tyrosine Decarboxylase

Entrapment of Tyrosine Decarboxylase (from Streptococcus faecalis,Fluka) was performed using 50 mM pH 5.5 acetate buffer and wasaccomplished by the method outlined above. The activity assay wasaccomplished using a 50:50 mixture of 2.5 mM solution of L-tyrosine(Aldrich, 99+%) in buffer and buffer. Total reaction volume for thisassay was either 40 mL (19.5 h data reported herein) or 100 mL (allother data reported). 2 mL aliquots of reaction mixture were removedfrom the reaction vessel for assay. The method used for this assay wasaddition of 1 mL of a 1M K2CO3 solution to the 2 mL aliquot, followed bymixing. To this was added 2 drops of a solution of picrylsulfonic acid(5% w/v aquesous solution of 2,4,6-trinitrobenzenesulfonic acid, Sigma).The mixture was mixed well. 2 mL of toluene were added to this mixture,the layers shaken well, and centrifuged. The toluene layer was removedand its UV-Vis spectrum collected. Where applicable, a 0.1 mM solutionof pyridoxal-5-phosphate monohydrate (98%, Aldrich) in buffer wassubstituted for the buffer solution in the assay mixture. Assaysperformed in the presence of cofactor were carried out in foil-coveredreaction vessels due to the sensitivity of pyridoxal-5-phosphate tolight.

3. Results of Enzyme Encapsulation and Assays

a) b-Glucosidase entrapment yielded active matrices which were assayedusing the synthetic substrate para-nitrophenyl-b-D-glucopyranoside,shown below. Enzymatic activity of matrix composites on the syntheticsubstrate results in cleavage of the glucosidic bond producing abathochromic shift in the spectral band. This shift permits monitoringof the cleavage process, as illustrated in FIG. 1.

The synthetic substrate para-nitrophenyl-b-D-glucopyranoside.

b) Penicillinase entrapment provided active matrices which were assayedusing the sodium salt of the synthetic substrate benzylpenicillin, shownbelow. Conversion of penicillin to penicilloic acid via rupture of theP-lactam ring may be monitored spectrophotometrically, as shown in FIG.2.

Substrate used for penicillinase activity assay, sodiumbenzylpenicillin.

Reproducibility of the measurements done for penicillinase was checkedby performing activity assay multiple times for the same matrix. Asshown in FIG. 3(a), good agreement is observed for multiple assaysperformed over six consecutive days. Likewise, running multiple matricesfrom the same preparation to check reproducibility of the matrixentrapment shows good agreement, as seen in FIG. 3(b).

Loading studies utilizing penicillinase matrices were performed in whichthe enzyme concentration was varied over a wide range to determine theoptimal enzyme concentration. The bar graph shown in FIG. 4 shows theeffects of varying enzyme concentration on the activity of the matrix.The highest percentage of activity observed as a function of enzymeentrapped within the matrix (selected from the five compositionsanalyzed) occurs for the lowest concentration of enzyme examined, asshown in FIG. 5.

Surface area effects were also examined utilizing penicillinasematrices. Ten identical monoliths were prepared and aged simultaneously.Five of these were assayed as whole monoliths (cast in 4.5 mL cuvettes)while another five matrices were coarsely crushed and then assayed. Thisqualitative examination of surface area effects revealed that anincrease in surface area does result in an increase in the enzymeactivity observed, as shown in FIGS. 6 and 7. It should be noted thatthere is no leaching of enzyme observed from either the whole or crushedmatrices, as determined by soaking the matrices in buffer solutionovernight and subsequently checking the activity of the soak solution.The reproducibility of the measurements for the assays shown in FIG. 6is quite reasonable, with the larger deviation in the crushed matricesattributable to the lack of control over particle size when breaking upthe samples. FIG. 7, showing the mean values for each measurement witherror bars, emphasizes the greater relative activity of the crushedmatrix samples.

The significant effect of changing surface area on the observed enzymeactivity prompted further investigation. Control over total surface areawas achieved by casting the sol containing penicillinase into varyingnumbers of cell culture plate wells (22.6 mm diameter). By varying theamount of sol cast into a given well, the total 4 mL of material permatrix could be spread out over a number of wells and the disks cast inthese wells could be recombined, after removal from the wells, forassay. Thus, the 4 mL of sol that constitutes one matrix preparationcould be cast into one or multiple wells to generate samples with known,varying surface areas. Surface area stated for a given matrix reflectsthe initial surface area of the gel when freshly cast, and does notattempt to correct for any shrinkage that occurred during aging. FIG.8(a) illustrates the difference in activity observed for matrices ofvarying surface areas. FIG. 8(b) shows the activity as a percentage ofthe penicillinase activity used in the preparation of the matrices.

c) Following entrapment of tyrosinase, the bifunctional activity of thisenzyme was found to complicate spectrophotometric assay of the matrixcomposite due to the variation in molar extinction coefficient of thedifferent species, and possible retention within the matrix. Tyrosinasepossesses both cresolase (conversion of phenols to diphenols) andcatecholase activity (conversion of diphenols to the correspondingquinone), as shown below. However, a qualitative analysis shows theconversion of the natural substrate L-tyrosine to L-dopa, which thenundergoes dehydrogenation to give dopaquinone. Dopaquinone is unstablein aqueous solutions and undergoes a Michealis rearrangement to form,among other products, a number of melanin precursors which eventuallypolymerize to produce pigments. This complicated reaction may befollowed qualitatively by monitoring a color change within the matrix.Although the L-tyrosine solution is, itself, colorless, thetyrosinase-containing matrices become noticeably darkened within onehour of contact with the substrate solution, suggesting formation ofproducts with subsequent retention by the matrix. A solution of L-dopain the presence of tyrosinase, likewise forms a gray-black precipitate.

d) Active Tyrosine Decarboxylase matrices were and assayed usingL-tyrosine as substrate. A complicating factor in the assay of entrappedTyrosine Decarboxylase was the unavailability of a directspectrophotometric method due to the equivalent molar extinctioncoefficients of substrate and product. The observation necessitated thedevelopment of an indirect assay provided by Phan et. al., App. Biochem.Biotech. 8:127 (1983). The results of an active Tyrosine Decarboxylasecomposite assay are shown in FIG. 9.

Longer aging times for Tyrosine Decarboxylase-containing matricesresulted in matrices for which no enzyme activity was observed withoutaddition of cofactor. Addition of cofactor, pyridoxal-5-phosphatemonohydrate (0.05 mM), to the assay mixture restored activity of theentrapped enzyme to varying degrees depending on the aging of themonolith. FIG. 10 shows activity assays for two 16 day old TyrosineDecarboxylase-containing matrices, one without cofactor present and onewith cofactor, and compares them to a matrix of the same compositionassayed after aging 19 h. The activity observed at 19 h without cofactorand at 16 d with cofactor present are nearly identical, whereas withoutthe presence of cofactor little, if any, activity is noted. For matricesaged 50 d a significant portion of the activity is retained in thepresence in cofactor, although some loss of activity is observed.

B. Matrix Optimization Studies

1. Matrix Preparation

The general synthetic technique used for preparation of the silica solwas addition of appropriate aliquots of the organically substitutedtrimethoxysilane, tetramethyl orthosilicate and 4 mM HCl solution to a25×150 mm test tube equipped with a stirbar. Total desired volume of solwas determined by the number of matrices to be prepared. The RSi(OCH₃)₃and TMOS reagents were combined in appropriate ratios to yield thedesired compositions. Reagent Source Tetramethylorthosilicate Aldrich,99+% (TMOS) Methyltrimethoxysilane Aldrich, 98% (MTMS)Ethyltrimethoxysilane Aldrich, 97+% (ETMS) TrimethoxypropylsilaneAldrich, 98% (TMPS) iso-Butyltrimethoxysilane Aldrich, 97% (i-BTMS)n-Butyltrimethoxysilane United Chemical (n-BTMS) Technologies, 95.3%Phenyltrimethoxysilane Aldrich, 97% (PTMS)

As with 100% TMOS matrices, the mixture is stirred until homogeneous(approximately 15 minutes). The test tube containing the sol is thentransferred to an ice bath and allowed to cool for 10 minutes. A 2 mLaliquot of sol is then transferred to another chilled test tube in anice bath and stirred. To this sol, 1 mL of chilled buffer solution(appropriate to the enzyme to be entrapped) is added, and stirred forca. 10s, followed by addition of 1 mL of chilled, buffered solutioncontaining the desired enzyme. The sol is swirled briefly, and thenpipetted into a 4.5 mL polystyrene cuvette (cell culture dishes werealso used for surface area study matrices). The cuvette opening issealed with Parafilm following gel formation (cell culture dish coverswere used for surface area study matrices). The gel is then allowed toage in the sealed container for a period of time ranging from 14 to 50days or more at temperatures ranging from 4° C. to room temperature.

2. Enzyme Encapsulation and Assays

Entrapment of Penicillinase (Type I from Bacillus cereus, lyophilizedpowder containing approx. 10% protein, Sigma) was performed as outlinedabove, using 50 mM pH 6.5 phosphate buffer. Penicillinase activity wasdetermined using 100 mL of a 3 mM solution of Penicillin G(Benzylpenicillin, sodium salt, Sigma) in buffer. 2 mL aliquots of thereaction solution were removed for assay and their UV-Vis spectrarecorded.

3. Results of Enzyme Encapsulation and Assays

Initial examination of which matrix compositions provided matricessuitable for the purposes of this study excluded then-butyltrimethoxysilane composition, as well as some of the higherratios of other RSi(OCH₃)₃ precursors, due to the failure of thesecompositions to form a gel that was appropriate for our intended uses.Compositions examined, and their reactivity relative to 100% TMOSmatrices are shown in Table 1. TABLE 1 Activity for given compositionsrelative to 100% TMOS. Composition Relative Activity MTMS:TMOS 10%MTMS:90% TMOS 108%  20% MTMS:80% TMOS 82% 30% MTMS:70% TMOS 92% 40%MTMS:60% TMOS 104%  50% MTMS:50% TMOS 112%* ETMS:TMOS 10% ETMS:90% TMOS99% 20% ETMS:80% TMOS 93% 30% ETMS:70% TMOS 99% TMPS:TMOS 10% TMPS:90%TMOS 100%  20% TMPS:80% TMOS 80% i-BTMS:TMOS 10% i-BTMS:90% TMOs 90% 20%i-BTMS:80% TMOs 75% PTMS:TMOS 10% PTMS:90% TMOS 94%*Indicates a composition in which slight enzyme leaching is observed atthe time of the assay. If insufficient aging is allowed, enzyme leachingis observed for methyltrimethoxysilane composition with MTMS contentgreater than 20%.

In addition, it was observed that as the matrices age the relativeactivity of the MTMS-containing matrices with respect to 100% TMOSdrops. When matrices from the same preparation are assayed after aging104 days at 4° C., the relative activity observed is shown in Table 2.TABLE 2 Enzyme activity relative to 100% TMOS for varyingMTMS-containing matrices aged 104 days. Composition Relative ActivityMTMS:TMOS 10% MTMS:90% TMOS 85% 20% MTMS:80% TMOS 78% 30% MTMS:70% TMOS85% 40% MTMS:60% TMOS 85% 50% MTMS:50% TMOS 103%**Indicates a composition in which no enzyme leaching is observed at thetime of this assay, although leaching is observed for shorter agingtime.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference and set forth in its entirety herein. In case of conflict, thepresent application, including any definitions herein, will control. Inaddition to the foregoing materials, the practice of the presentinvention may employ in part, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning a Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning. Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat.No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higginseds. 1984); Transcription and Translation (B. D. Hames & S. J. Higginseds. 1984); Culture Of Animal Cells (R. I. Fresliney, Alan R. Liss,Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,A Practical Guide To Molecular Cloning (1984); the treatise Methods InEnzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Methods in Enzymologv, Vols. 154 and 155 (Wu et al.eds.), Immunochemical Methods in Cell and Molecular Biology (Mayer andWalker, eds., Academic Press, London, +987); Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986);Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986), all of which references are herebyincorporated by reference to the same extent as the other referencesspecified herein.

The specification and examples should be considered exemplary only withthe true scope and spirt of the invention suggested by the followingclaims.

1-138. (canceled)
 139. A method of toxicology testing, comprising: a.encapsulating at least one reaction center in a silica-based sol-gelmatrix; b. interacting a compound with said matrix; and, c. evaluatingfor any products of said compound resulting from conversion of saidcompound by said reaction center, wherein production of any cytotoxic ormutagenic products indicates that said compound may be toxic to asubject upon administration.
 140. The method of claim 139, wherein saidreaction center comprises an enzyme that is located in the liver of amammal.
 141. The method of claim 139, wherein said reaction center is anenzyme prepared by recombinant methods.
 142. The method of claim 139,wherein said reaction center is cell-free.
 143. The method of claim 140,wherein said mammal is one of the following: a pig or a human.